Antigen binding proteins to proprotein convertase subtilisin kexin type 9 (pcsk9)

ABSTRACT

Antigen binding proteins that interact with Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9) are described. Methods of treating hypercholesterolemia and other disorders by administering a pharmaceutically effective amount of an antigen binding protein to PCSK9 are described. Methods of detecting the amount of PCSK9 in a sample using an antigen binding protein to PCSK9 are described.

RELATED APPLICATIONS

This application is a divisional and claims priority to U.S.Non-Provisional application Ser. No. 12/197,093, filed Aug. 22, 2008,and U.S. Provisional Application Ser. No. 61/086,133, filed Aug. 4,2008, Ser. No. 60/957,668, filed Aug. 23, 2007, Ser. No. 61/008,965,filed Dec. 21, 2007, and Ser. No. 61/010,630, filed Jan. 9, 2008, herebyincorporated by reference in their entireties.

SEQUENCE LISTING AND TABLES IN ELECTRONIC FORMAT

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledSequenceListing-APMOL-003D1.txt, created Sep. 28, 2011, last saved Sep.30, 2011 which is 296,708 bytes in size. The information in theelectronic format of the Sequence Listing is incorporated herein byreference in its entirety. The present application is being filed alongwith a collection of Tables in electronic format. The collection ofTables is provided as a file entitled Table_(—)35-1-4_APMOL-003D1.txt,created and last saved on Sep. 30, 2011, which is 2,024,359 bytes insize. The information in the electronic format of the collection ofTables is incorporated herein by reference in its entirety.

LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20120020976A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

FIELD OF THE INVENTION

The present invention relates to antigen binding proteins that bind toproprotein convertase subtilisin kexin type 9 (PCSK9) and methods ofusing and making the antigen binding proteins.

BACKGROUND OF VARIOUS EMBODIMENTS

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a serineprotease involved in regulating the levels of the low densitylipoprotein receptor (LDLR) protein (Horton et al., 2007; Seidah andPrat, 2007). In vitro experiments have shown that adding PCSK9 to HepG2cells lowers the levels of cell surface LDLR (Benjannet et al., 2004;Lagace et al., 2006; Maxwell et al., 2005; Park et al., 2004).Experiments with mice have shown that increasing PCSK9 protein levelsdecreases levels of LDLR protein in the liver (Benjannet et al., 2004;Lagace et al., 2006; Maxwell et al., 2005; Park et al., 2004), whilePCSK9 knockout mice have increased levels of LDLR in the liver (Rashidet al., 2005). Additionally, various human PCSK9 mutations that resultin either increased or decreased levels of plasma LDL have beenidentified (Kotowski et al., 2006; Zhao et al., 2006). PCSK9 has beenshown to directly interact with the LDLR protein, be endocytosed alongwith the LDLR, and co-immunofluoresce with the LDLR throughout theendosomal pathway (Lagace et al., 2006). Degradation of the LDLR byPCSK9 has not been observed and the mechanism through which it lowersextracellular LDLR protein levels is uncertain.

PCSK9 is a prohormone-proprotein convertase in the subtilisin (S8)family of serine proteases (Seidah et al., 2003). Humans have nineprohormone-proprotein convertases that can be divided between the S8Aand S8B subfamilies (Rawlings et al., 2006). Furin, PC1/PC3, PC2, PACE4,PC4, PC5/PC6 and PC7/PC8/LPC/SPC7 are classified in subfamily S8B.Crystal and NMR structures of different domains from mouse furin and PC1reveal subtilisin-like pro- and catalytic domains, and a P domaindirectly C-terminal to the catalytic domain (Henrich et al., 2003;Tangrea et al., 2002). Based on the amino acid sequence similaritywithin this subfamily, all seven members are predicted to have similarstructures (Henrich et al., 2005). SKI-1/S1P and PCSK9 are classified insubfamily S8A. Sequence comparisons with these proteins also suggest thepresence of subtilisin-like pro- and catalytic domains (Sakai et al.,1998; Seidah et al., 2003; Seidah et al., 1999). In these proteins theamino acid sequence C-terminal to the catalytic domain is more variableand does not suggest the presence of a P domain.

Prohormone-proprotein convertases are expressed as zymogens and theymature through a multi step process. The function of the pro-domain inthis process is two-fold. The pro-domain first acts as a chaperone andis required for proper folding of the catalytic domain (Ikemura et al.,1987). Once the catalytic domain is folded, autocatalysis occurs betweenthe pro-domain and catalytic domain. Following this initial cleavagereaction, the pro-domain remains bound to the catalytic domain where itthen acts as an inhibitor of catalytic activity (Fu et al., 2000). Whenconditions are correct, maturation proceeds with a second autocatalyticevent at a site within the pro-domain (Anderson et al., 1997). Afterthis second cleavage event occurs the pro-domain and catalytic domaindissociate, giving rise to an active protease.

Autocatalysis of the PCSK9 zymogen occurs between Gln152 and Ser153(VFAQ|SIP) (Naureckiene et al., 2003), and has been shown to be requiredfor its secretion from cells (Seidah et al., 2003). A secondautocatalytic event at a site within PCSK9's pro-domain has not beenobserved. Purified PCSK9 is made up of two species that can be separatedby non-reducing SDS-PAGE; the pro-domain at 17 Kd, and the catalyticplus C-terminal domains at 65 Kd. PCSK9 has not been isolated withoutits inhibitory pro-domain, and measurements of PCSK9's catalyticactivity have been variable (Naureckiene et al., 2003; Seidah et al.,2003).

SUMMARY OF VARIOUS EMBODIMENTS

In some embodiments, the invention comprises an antigen binding proteinto PCSK9.

In some aspects, the invention comprises an isolated antigen bindingprotein that binds PCSK9 comprising: A) one or more heavy chaincomplementary determining regions (CDRHs) selected from the groupconsisting of: (i) a CDRH1 from a CDRH1 in a sequence selected from thegroup consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53,48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83,69, 81, and 60; (ii) a CDRH2 from a CDRH2 in a sequence selected fromthe group consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51,53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78,83, 69, 81, and 60; (iii) a CDRH3 from a CDRH3 in a sequence selectedfrom the group consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52,51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77,78, 83, 69, 81, and 60; and (iv) a CDRH of (i), (ii), and (iii) thatcontains one or more amino acid substitutions, deletions or insertionsof no more than 4 amino acids; B) one or more light chain complementarydetermining regions (CDRLs) selected from the group consisting of: (i) aCDRL1 from a CDRL1 in a sequence selected from the group consisting ofSEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46; (ii) aCDRL2 from a CDRL2 in a sequence selected from the group consisting ofSEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46; (iii) aCDRL3 from a CDRL3 in a sequence selected from the group consisting ofSEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46; and (iv)a CDRL of (i), (ii) and (iii) that contains one or more amino acidsubstitutions, deletions or insertions of no more than 4 amino acids; orC) one or more heavy chain CDRHs of A) and one or more light chain CDRLsof B). In some embodiments, the isolated antigen binding proteincomprises at least one CDRH of A) and at least one CDRL of B). In someembodiments, the isolated antigen binding protein comprises at least twoCDRH of A) and at least two CDRL of B). In some embodiments, theisolated antigen binding protein comprises said CDRH1, CDRH2, CDRH3,CDRL1, CDRL2 and CDRL3. In some embodiments, the CDRH of A) is selectedfrom at least one of the group consisting of: (i) a CDRH1 amino acidsequence selected from the CDRH1 in a sequence selected from the groupconsisting of SEQ ID NO: 67, 79, 89, and 49; (ii) a CDRH2 amino acidsequence selected from the CDRH2 in a sequence selected from the groupconsisting of SEQ ID NO: 67, 79, 89, and 49; (iii) a CDRH3 amino acidsequence selected from the CDRH3 in a sequence selected from the groupconsisting of SEQ ID NO: 67, 79, 89, and 49; and (iv) a CDRH of (i),(ii) and (iii) that contains one or more amino acid substitutions,deletions or insertions of no more than 2 amino acids. In addition, theCDRL of B) is selected from at least one of the group consisting of: (i)a CDRL1 amino acid sequence selected from the CDRL1 in a sequenceselected from the group consisting of SEQ ID NO: 12, 35, 32, and 23;(ii) a CDRL2 amino acid sequence selected from the CDRL2 in a sequenceselected from the group consisting of SEQ ID NO: 12, 35, 32, and 23;(iii) a CDRL3 amino acid sequence selected from the CDRL3 in a sequenceselected from the group consisting of SEQ ID NO: 12, 35, 32, and 23; and(iv) a CDRL of (i), (ii) and (iii) that contains one or more amino acidsubstitutions, deletions or insertions of no more than 2 amino acids; orC) one or more heavy chain CDRHs of A) and one or more light chain CDRLsof B. In some embodiments, the CDRH of A) is selected from at least oneof the group consisting of: (i) a CDRH1 amino acid sequence of the CDRH1amino acid sequence in SEQ ID NO: 67; (ii) a CDRH2 amino acid sequenceof the CDRH2 amino acid sequence in SEQ ID NO: 67; (iii) a CDRH3 aminoacid sequence of the CDRH3 amino acid sequence in SEQ ID NO: 67; and(iv) a CDRH of (i), (ii) and (iii) that contains one or more amino acidsubstitutions, deletions or insertions of no more than 2 amino acids;said CDRL of B) is selected from at least one of the group consistingof: (i) a CDRL1 amino acid sequence of the CDRL1 amino acid sequence inSEQ ID NO: 12; (ii) a CDRL2 amino acid sequence of the CDRL2 amino acidsequence in SEQ ID NO: 12; (iii) a CDRL3 amino acid sequence of theCDRL3 amino acid sequence in SEQ ID NO: 12; and (iv) a CDRL of (i), (ii)and (iii) that contains one or more amino acid substitutions, deletionsor insertions of no more than 2 amino acids; or C) one or more heavychain CDRHs of A) and one or more light chain CDRLs of B). In someembodiments, the antigen binding protein comprises A) a CDRH1 of theCDRH1 sequence in SEQ ID NO: 67, a CDRH2 of the CDRH2 sequence in SEQ IDNO: 67, and a CDRH3 of the CDRH3 sequence in SEQ ID NO: 67, and B) aCDRL1 of the CDRL1 sequence in SEQ ID NO: 12, a CDRL2 of the CDRL2sequence in SEQ ID NO: 12, and a CDRL3 of the CDRL3 sequence in SEQ IDNO: 12. In some embodiments, the antigen binding protein comprises aheavy chain variable region (VH) having at least 80% sequence identitywith an amino acid sequence selected from the group consisting of SEQ IDNO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50,91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60, and/or alight chain variable region (VL) having at least 80% sequence identitywith an amino acid sequence selected from the group consisting of SEQ IDNO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28,30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. In someembodiments, the VH has at least 90% sequence identity with an aminoacid sequence selected from the group consisting of SEQ ID NO: 74, 85,71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62,89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60, and/or the VL has atleast 90% sequence identity with an amino acid sequence selected fromthe group consisting of SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40,42, 44, and 46. In some embodiments, the VH is selected from the groupconsisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54,55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81,and 60, and/or the VL is selected from the group consisting of SEQ IDNO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28,30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46.

In some aspects, the invention comprises an isolated antigen bindingprotein that specifically binds to an epitope that is bound by any ofthe ABPs disclosed herein.

In some aspects, the invention comprises an isolated antigen bindingprotein that binds PCSK9, wherein the antigen binding protein comprises:A) one or more heavy chain CDRs (CDRHs) selected from at least one ofthe group consisting of: (i) a CDRH1 with at least 80% sequence identityto a CDRH1 in one of the sequences selected from the group consisting ofSEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49,57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60; (ii)a CDRH2 with at least 80% sequence identity to a CDRH2 in one of thesequences selected from the group consisting of SEQ ID NO: 74, 85, 71,72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89,65, 79, 80, 76, 77, 78, 83, 69, 81, and 60; and (iii) a CDRH3 with atleast 80% sequence identity to a CDRH3 in one of the sequences selectedfrom the group consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52,51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77,78, 83, 69, 81, and 60; B) one or more light chain CDRs (CDRLs) selectedfrom at least one of the group consisting of: (i) a CDRL1 with at least80% sequence identity to a CDRL1 in one of the sequences selected fromthe group consisting of SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40,42, 44, and 46; (ii) a CDRL2 with at least 80% sequence identity to aCDRL2 in one of the sequences selected from the group consisting of SEQID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26,28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46; and (iii) aCDRL3 with at least 80% sequence identity to a CDRL3 in one of thesequences selected from the group consisting of SEQ ID NO: 5, 7, 9, 10,12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33,35, 36, 37, 38, 39, 40, 42, 44, and 46; or C) one or more heavy chainCDRHs of A) and one or more light chain CDRLs of B). In someembodiments, the antigen binding protein comprises: A) one or more CDRHsselected from at least one of the group consisting of: (i) a CDRH1 withat least 90% sequence identity to a CDRH1 in one of the sequencesselected from the group consisting of SEQ ID NO: 74, 85, 71, 72, 67, 87,58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80,76, 77, 78, 83, 69, 81, and 60; (ii) a CDRH2 with at least 90% sequenceidentity to a CDRH2 in one of the sequences selected from the groupconsisting of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54,55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81,and 60; and (iii) a CDRH3 with at least 90% sequence identity to a CDRH3in one of the sequences selected from the group consisting of SEQ ID NO:74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91,64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60; B) one or moreCDRLs selected from at least one of the group consisting of: (i) a CDRL1with at least 90% sequence identity to a CDRL1 in one of the sequencesselected from the group consisting of SEQ ID NO: 5, 7, 9, 10, 12, 13,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36,37, 38, 39, 40, 42, 44, and 46; (ii) a CDRL2 with at least 90% sequenceidentity to a CDRL2 in one of the sequences selected from the groupconsisting of SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44,and 46; and (iii) a CDRL3 with at least 90% sequence identity to a CDRL3in one of the sequences selected from the group consisting of SEQ ID NO:5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30,31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46; or C) one or moreheavy chain CDRHs of A) and one or more light chain CDRLs of B).

In some aspects, the invention comprises an isolated antigen bindingprotein that binds PCSK9, the antigen binding protein comprises: A) aheavy chain complementary determining region (CDRH) selected from atleast one of the group consisting of: (i) a CDRH3 selected from theCDRH3 within the sequences selected from the group consisting of SEQ IDNOs: 67, 79, and 49, (ii) a CDRH3 that differs in amino acid sequencefrom the CDRH3 of (i) by an amino acid addition, deletion orsubstitution of not more than two amino acids; and (iii)X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄ (SEQ ID NO: 404), wherein X₁ isselected from the group consisting of D, A, R, and not amino acid, X₂ isselected from the group consisting of Y, I, G, and no amino acid, X₃ isselected from the group consisting of D, A, G, and no amino acid, X₄ isselected from the group consisting of F, A, L, and no amino acid, X₅ isselected from the group consisting of W, L, A, and no amino acid, X₆ isselected from the group consisting of S, Y, A, and no amino acid, X₇ isselected from the group consisting of A, Y, R, and no amino acid, X₈ isselected from the group consisting of Y, P, and no amino acid, X₉ isselected from the group consisting of Y, G, and no amino acid, X₁₀ isselected from the group consisting of D, G, and no amino acid, X₁₁ isselected from the group consisting of A, M, and no amino acid, X₁₂ isselected from the group consisting of F, D, and no amino acid, X₁₃ isselected from the group consisting of D, V, and no amino acid, X₁₄ isselected from the group consisting of V and no amino acid; B) a lightchain complementary determining region (CDRL) selected from at least oneof the group consisting of: (i) a CDRL3 selected from the CDRL3 withinthe sequences selected from the group consisting of SEQ ID NOs: 12, 35,and 23, (ii) a CDRL3 that differs in amino acid sequence from the CDRL3of (i) by an amino acid addition, deletion or substitution of not morethan two amino acids; and (iii) a CDRL3 amino acid sequence selectedfrom the group consisting of: X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁ (SEQ ID NO: 405),wherein X₁ is selected from the group consisting of Q and G, X₂ isselected from the group consisting of S, T, A, and no amino acid, X₃ isselected from the group consisting of Y, no amino acid, and W, X₄ isselected from the group consisting of D and no amino acid, X₅ isselected from the group consisting of S and no amino acid, X₆ isselected from the group consisting of S and no amino acid, X₇ isselected from the group consisting of L, T, and no amino acid, X₈ isselected from the group consisting of no amino acid, A, and S, X₉ isselected from the group consisting of no amino acid, G, A, and V, X₁₀ isselected from the group consisting of no amino acid, S, Y, and V, X₁₁ isselected from the group consisting of no amino acid and V.

In some aspects, the invention comprises an isolated antigen bindingprotein comprising a light chain having the amino acid sequence selectedfrom the group consisting of: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42,44, 46, and some combination thereof.

In some embodiments, the antigen binding protein specifically binds toan epitope that is bound by at least one of the antigen binding proteinsdisclosed herein. In some embodiments, the isolated antigen bindingprotein further comprises a heavy chain having the amino acid sequenceselected from the group consisting of: 74, 85, 71, 72, 67, 87, 58, 52,51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77,78, 83, 69, 81, 60, and some combination thereof. In some embodiments,the amino acid sequence of the ABP is selected from the group consistingof SEQ ID NO: 12, 35, 23, and some combination thereof. In someembodiments, the heavy chain of the ABP comprises a CDRH3 of SEQ ID NO:67, a CDRH2 of SEQ ID NO: 67, and a CDRH1 of SEQ ID NO:67, and saidlight chain comprises a CDRL3 of SEQ ID NO: 12, a CDRL2 of SEQ ID NO:12, and a CDRL1 of SEQ ID NO: 12. In some embodiments, the isolatedantigen binding protein is a monoclonal antibody, a polyclonal antibody,a recombinant antibody, a human antibody, a humanized antibody, achimeric antibody, a multispecific antibody, or an antibody fragmentthereof. In some embodiments, the isolated antigen binding protein is aFab fragment, a Fab′ fragment, a F(ab′)₂ fragment, a Fv fragment, adiabody, or a single chain antibody molecule. In some embodiments, theisolated antigen binding protein is a human antibody. In someembodiments, the isolated antigen binding protein is a monoclonalantibody. In some embodiments, the isolated antigen binding protein isof the IgG1-, IgG2- IgG3- or IgG4-type. In some embodiments, theisolated antigen binding protein is of the IgG4- or IgG2-type. In someembodiments, the isolated antigen binding protein is coupled to alabeling group. In some embodiments, the isolated antigen bindingprotein competes for binding to PCSK9 with an antigen binding proteindescribed herein. In some embodiments, the isolated antigen bindingprotein is a monoclonal antibody, a polyclonal antibody, a recombinantantibody, a human antibody, a humanized antibody, a chimeric antibody, amultispecific antibody, or an antibody fragment thereof. In someembodiments, the isolated antigen binding protein is a Fab fragment, aFab′ fragment, a F(ab′)₂ fragment, a Fv fragment, a diabody, or a singlechain antibody molecule. In some embodiments, the isolated antigenbinding protein is coupled to a labeling group. In some embodiments, theisolated antigen binding protein reduces binding of PCSK9 to LDLR. Insome embodiments, the isolated antigen binding protein the antigenbinding protein decreases an amount of LDL present in a subject whenadministered to the subject. In some embodiments, the isolated antigenbinding protein decreases an amount of serum cholesterol present in asubject when administered to the subject. In some embodiments, theisolated antigen binding protein increases an amount of LDLR present ina subject when administered to the subject.

In some aspects, the invention comprises a vector comprising a nucleicacid molecule as described herein. In some embodiments, the inventioncomprises a host cell comprising a nucleic acid molecule as describedherein.

In some aspects, the invention comprises an isolated antigen bindingprotein that competes for binding to PCSK9 with an antigen bindingprotein disclosed herein.

In some aspects, the invention comprises a nucleic acid moleculeencoding the antigen binding protein according disclosed herein.

In some aspects, the invention comprises a pharmaceutical compositioncomprising at least one antigen binding protein described herein.

In some aspects, the invention comprises a method for treating orpreventing a condition associated with elevated serum cholesterol levelsin a patient, comprising administering to a patient in need thereof aneffective amount of at least one isolated antigen binding proteindisclosed herein.

In some aspects, the invention comprises a method of inhibiting bindingof PCSK9 to LDLR in a subject comprising administering an effectiveamount of at least one antigen binding protein disclosed herein.

In some aspects, the invention comprises an antigen binding protein thatselectively binds to PCSK9, wherein the antigen binding protein binds toPCSK9 with a K_(d) that is smaller than 100 pM.

In some aspects, the invention comprises a method for treating orpreventing a condition associated with elevated serum cholesterol levelsin a subject, the method comprising administering to a subject in needthereof an effective amount of at least one isolated antigen bindingprotein disclosed herein simultaneously or sequentially with an agentthat elevates the availability of LDLR protein.

In some aspects, the invention comprises a method of lowering serumcholesterol level in a subject, the method comprising administering to asubject an effective amount of at least one isolated antigen bindingprotein as disclosed herein.

In some aspects, the invention comprises a method of lowering serumcholesterol level in a subject, the method comprising administering to asubject an effective amount of at least one isolated antigen bindingprotein as disclosed herein, simultaneously or sequentially with anagent that elevates the availability of LDLR protein.

In some aspects, the invention comprises a method of increasing LDLRprotein level in a subject, the method comprising administering to asubject an effective amount of at least one isolated antigen bindingprotein as disclosed herein.

In some aspects, the invention comprises a method of increasing LDLRprotein levels in a subject, the method comprising administering to asubject an effective amount of at least one isolated antigen bindingprotein as disclosed herein simultaneously or sequentially with an agentthat elevates the availability of LDLR protein.

In some aspects, the invention comprises a pharmaceutical compositioncomprising an ABP as disclosed herein and an agent that elevates theavailability of LDLR protein levels. In some embodiments, the agent thatelevates the availability of LDLR protein comprises a statin. In someembodiments, the statin is selected from the group consisting ofatorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin,pitavastatin, pravastatin, rosuvastatin, simvastatin, and somecombination thereof.

In some aspect, the invention comprises a method of making the antigenbinding protein as described herein, comprising the step of preparingsaid antigen binding protein from a host cell that secretes said antigenbinding protein.

In some aspect, the invention comprises a pharmaceutical compositioncomprising at least one antigen binding protein as described herein anda pharmaceutically acceptable excipient. In some embodiments, thepharmaceutical composition further comprises an additional active agent.In some embodiments, said additional active agent is selected from thegroup consisting of a radioisotope, radionuclide, a toxin, or atherapeutic and a chemotherapeutic group.

In some aspects, the invention comprises a method for treating orpreventing a condition associated with an elevated serum cholesterollevel in a patient. The method comprises administering to a patient inneed thereof an effective amount of at least one isolated antigenbinding protein as disclosed herein. In some embodiments, the conditionis hypercholesterolemia.

In some aspects, the invention comprises a method of inhibiting bindingof PCSK9 to LDLR in a patient comprising administering an effectiveamount of at least one antigen binding protein according as describedherein.

In some aspect, the invention comprises an antigen binding protein thatbinds to PCSK9 with a K_(d) that is smaller than 100 pM. In someembodiments, the antigen binding protein binds with a K_(d) that issmaller than 10 pM. In some embodiments, the antigen binding proteinbinds with a K_(d) that is less than 5 pM.

In some aspects, the invention comprises a method for treating orpreventing a condition associated with elevated serum cholesterol levelsin a subject, said method comprising administering to a subject in needthereof an effective amount of at least one isolated antigen bindingprotein described herein simultaneously or sequentially with an agentthat elevates the availability of LDLR protein. In some embodiments, theagent that elevates the availability of LDLR protein comprises a statin.In some embodiments, the statin is selected from the group consisting ofatorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin,pitavastatin, pravastatin, rosuvastatin, simvastatin, and somecombination thereof.

In some aspects, the invention comprises a method of lowering the serumcholesterol level in a subject. The method comprises administering to asubject an effective amount of at least one isolated antigen bindingprotein as described herein.

In some aspects, the invention comprises a method of lowering serumcholesterol levels in a subject comprising administering to a subject aneffective amount of at least one isolated antigen binding protein, asdescribed herein, simultaneously or sequentially with an agent thatelevates the availability of LDLR protein. In some embodiments, theagent that elevates the availability of LDLR protein comprises a statin.In some embodiments, the statin is selected from the group consisting ofatorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin,pitavastatin, pravastatin, rosuvastatin, simvastatin, and somecombination thereof.

In some aspects, the invention comprises a method of increasing LDLRprotein levels in a subject by administering to a subject an effectiveamount of at least one isolated antigen binding protein as providedherein.

In some aspects, the invention comprises a method of increasing LDLRprotein levels in a subject by administering to a subject an effectiveamount of at least one isolated antigen binding protein, as describedherein, simultaneously or sequentially with an agent that elevates theavailability of LDLR protein. In some embodiments, the agent thatelevates the availability of LDLR protein levels comprises a statin. Insome embodiments, the statin is selected from the group consisting ofatorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin,pitavastatin, pravastatin, rosuvastatin, simvastatin, and somecombination thereof.

In some aspects, the invention comprises a neutralizing antibody thatbinds to PCSK9 and reduces a low density lipoprotein receptor (LDLR)lowering effect of PCSK9 on LDLR. In some embodiments, the antibodyspecifically binds to PCSK9. In some embodiments, the antibody binds tothe catalytic domain of PCSK9. In some embodiments, the antibody bindsto an epitope within residues 31-447 of SEQ ID NO: 3. In someembodiments, the antibody binds to PCSK9 having an amino acid sequencethat is at least 90% identical to SEQ ID NO: 3.

In some aspects, the invention comprises a neutralizing antigen bindingprotein that binds to PCSK9, wherein the antigen binding protein bindsto PCSK9 at a location within residues 31-447 of SEQ ID NO: 3. In someembodiments, when the antigen binding protein is bound to PCSK9, theantibody is positioned 8 angstroms or less from at least one of thefollowing residues of PCSK9: S153, I154, P155, R194, D238, A239, I369,S372, D374, C375, T377, C378, F379, V380, S381, W156, N157, L158, E159,H193, E195, H229, R237, G240, K243, D367, I368, G370, A371, S373, S376,Q382, W72, F150, A151, Q152, T214, R215, F216, H217, A220, S221, K222,S225, H226, C255, Q256, G257, K258, N317, F318, T347, L348, G349, T350,L351, E366, D367, D374, V380, S381, Q382, S383, G384, K69, D70, P71,S148, V149, D186, T187, E211, D212, G213, R218, Q219, C223, D224, G227,H229, L253, N254, G259, P288, A290, G291, G316, R319, Y325, V346, G352,T353, G365, I368, I369, S372, S373, C378, F379, T385, S386, Q387, S153,S188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237,D238, K243, S373, D374, S376, T377, F379, I154, T187, H193, E195, I196,M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375,or C378. In some embodiments, the antibody is positioned 8 angstroms orless from at least one of the following residues of PCSK9: S153, I154,P155, R194, D238, A239, I369, S372, D374, C375, T377, C378, F379, V380,S381, W156, N157, L158, E159, H193, E195, H229, R237, G240, K243, D367,I368, G370, A371, S373, S376, or Q382. In some embodiments, the antibodyis positioned 5 angstroms or less from at least one of the followingresidues of PCSK9: S153, I154, P155, R194, D238, A239, I369, S372, D374,C375, T377, C378, F379, V380, or S381. In some embodiments, the antibodyis positioned 5 angstroms or less from at least two of the followingresidues of PCSK9: S153, I154, P155, R194, D238, A239, I369, S372, D374,C375, T377, C378, F379, V380, or S381. In some embodiments, the antibodyis 5 angstroms or less from at least four of the following residues ofPCSK9: S153, I154, P155, R194, D238, A239, I369, S372, D374, C375, T377,C378, F379, V380, or S381. In some embodiments, the antibody ispositioned 8 angstroms or less from at least one of the followingresidues of PCSK9: W72, F150, A151, Q152, T214, R215, F216, H217, A220,S221, K222, S225, H226, C255, Q256, G257, K258, N317, F318, T347, L348,G349, T350, L351, E366, D367, D374, V380, S381, Q382, S383, G384, K69,D70, P71, S148, V149, D186, T187, E211, D212, G213, R218, Q219, C223,D224, G227, H229, L253, N254, G259, P288, A290, G291, G316, R319, Y325,V346, G352, T353, G365, I368, I369, S372, S373, C378, F379, T385, S386,or Q387. In some embodiments, the antibody is positioned 5 angstroms orless from at least one of the following residues of PCSK9: W72, F150,A151, Q152, T214, R215, F216, H217, A220, S221, K222, S225, H226, C255,Q256, G257, K258, N317, F318, T347, L348, G349, T350, L351, E366, D367,D374, V380, S381, Q382, S383, or G384. In some embodiments, the antibodyis positioned 5 angstroms or less from at least two of the followingresidues of PCSK9: W72, F150, A151, Q152, T214, R215, F216, H217, A220,S221, K222, S225, H226, C255, Q256, G257, K258, N317, F318, T347, L348,G349, T350, L351, E366, D367, D374, V380, S381, Q382, S383, or G384. Insome embodiments, the antibody is positioned 5 angstroms or less from atleast four of the following residues of PCSK9: W72, F150, A151, Q152,T214, R215, F216, H217, A220, S221, K222, S225, H226, C255, Q256, G257,K258, N317, F318, T347, L348, G349, T350, L351, E366, D367, D374, V380,S381, Q382, S383, or G384. In some embodiments, the antibody ispositioned 8 angstroms or less from at least one of the followingresidues of PCSK9: S153, S188, I189, Q190, S191, D192, R194, E197, G198,R199, V200, D224, R237, D238, K243, S373, D374, S376, T377, F379, I154,T187, H193, E195, I196, M201, V202, C223, T228, S235, G236, A239, G244,M247, I369, S372, C375, or C378. In some embodiments, the antibody ispositioned 5 angstroms or less from at least one of the followingresidues of PCSK9: S153, S188, I189, Q190, S191, D192, R194, E197, G198,R199, V200, D224, R237, D238, K243, S373, D374, S376, T377, or F379. Insome embodiments, the antibody is positioned 5 angstroms or less from atleast two of the following residues of PCSK9: S153, S188, I189, Q190,S191, D192, R194, E197, G198, R199, V200, D224, R237, D238, K243, S373,D374, S376, T377, or F379. In some embodiments, the antibody ispositioned 5 angstroms or less from at least four of the followingresidues of PCSK9: S153, S188, I189, Q190, S191, D192, R194, E197, G198,R199, V200, D224, R237, D238, K243, S373, D374, S376, T377, or F379.

In some aspects, the invention comprises a neutralizing antibody thatbinds to PCSK9, wherein the antibody binds to PCSK9 and reduces thelikelihood that PCSK9 binds to LDLR.

In some embodiments, an antibody or antigen binding molecule that bindsto PCSK9 is contemplated. The antibody binds to PCSK9 at a locationwithin residues 31-447 of SEQ ID NO: 3. In some embodiments, theantibody or antigen binding molecule, when bound to PCSK9, is positioned8 angstroms or less from at least one of the following residues ofPCSK9: S153, I154, P155, R194, D238, A239, I369, S372, D374, C375, T377,C378, F379, V380, S381, W156, N157, L158, E159, H193, E195, H229, R237,G240, K243, D367, I368, G370, A371, S373, S376, Q382, W72, F150, A151,Q152, T214, R215, F216, H217, A220, S221, K222, S225, H226, C255, Q256,G257, K258, N317, F318, T347, L348, G349, T350, L351, E366, D367, D374,V380, S381, Q382, S383, G384, K69, D70, P71, S148, V149, D186, T187,E211, D212, G213, R218, Q219, C223, D224, G227, H229, L253, N254, G259,P288, A290, G291, G316, R319, Y325, V346, G352, T353, G365, I368, I369,S372, S373, C378, F379, T385, S386, Q387, S153, S188, I189, Q190, S191,D192, R194, E197, G198, R199, V200, D224, R237, D238, K243, S373, D374,S376, T377, F379, I154, T187, H193, E195, I196, M201, V202, C223, T228,S235, G236, A239, G244, M247, I369, S372, C375, or C378.

In some embodiments, an isolated antibody or antigen binding moleculethat blocks an antibody to PCSK9 from binding within 8 angstroms of aresidue of PCSK9 is provided. In some embodiments the residue of PCSK9is selected from at least one of the following PCSK9 residues: S153,I154, P155, R194, D238, A239, I369, S372, D374, C375, T377, C378, F379,V380, S381, W156, N157, L158, E159, H193, E195, H229, R237, G240, K243,D367, I368, G370, A371, S373, S376, Q382, W72, F150, A151, Q152, T214,R215, F216, H217, A220, S221, K222, S225, H226, C255, Q256, G257, K258,N317, F318, T347, L348, G349, T350, L351, E366, D367, D374, V380, S381,Q382, S383, G384, K69, D70, P71, S148, V149, D186, T187, E211, D212,G213, R218, Q219, C223, D224, G227, H229, L253, N254, G259, P288, A290,G291, G316, R319, Y325, V346, G352, T353, G365, I368, I369, S372, S373,C378, F379, T385, S386, Q387, S153, S188, I189, Q190, S191, D192, R194,E197, G198, R199, V200, D224, R237, D238, K243, S373, D374, S376, T377,F379, I154, T187, H193, E195, I196, M201, V202, C223, T228, S235, G236,A239, G244, M247, I369, S372, C375, or C378.

In some embodiments, an isolated antibody or antigen binding moleculethat binds to PCSK9 at a location that overlaps with a location thatLDLR binds to PCSK9 is provided. In some embodiments, the location thatLDLR binds to PCSK9 includes at least one amino acid residue selectedfrom the group consisting of: S153, I154, P155, R194, D238, A239, I369,S372, D374, C375, T377, C378, F379, V380, and S381.

In some embodiments, an isolated antibody or antigen binding moleculethat binds to PCSK9 is provided. In some embodiments, the antibody orantigen binding molecule reduces the likelihood that EGFa will bind toPCSK9 within 8 angstroms of at least one of the following residues onPCSK9: S153, I154, P155, R194, D238, A239, I369, S372, D374, C375, T377,C378, F379, V380, S381, W156, N157, L158, E159, H193, E195, H229, R237,G240, K243, D367, I368, G370, A371, S373, S376, or Q382.

In some embodiments, an antibody, antigen binding protein, or antigenbinding molecule that binds to a surface of PCSK9 that overlaps with asurface that EGFa binds, Ab 21B12 binds, and/or 31H4 binds is provided.In some embodiments, an antibody, antigen binding protein, or antigenbinding molecule that binds to PCSK9 in a manner that is similar to thatdepicted in the figures is provided.

In some embodiments, the above embodiments are neutralizing antibodiesor antigen binding proteins. In some embodiments, the antigen bindingprotein is not LDLR or a fragment thereof (such as EGFa).

In some aspects, the invention comprises an isolated neutralizingantibody, wherein when the antibody is bound to PCSK9, the antibody ispositioned 8 angstroms or less from at least one of the followingresidues of PCSK9: T468, R469, M470, A471, T472, R496, R499, E501, A502,Q503, R510, H512, F515, P540, P541, A542, E543, H565, W566, E567, V568,E569, R592, E593, S465, G466, P467, A473, I474, R476, G497, E498, M500,G504, K506, L507, V508, A511, N513, A514, G516, V536, T538, A539, A544,T548, D570, L571, H591, A594, S595, and H597 of SEQ ID NO: 3. In someembodiments, the antibody is positioned 5 angstroms or less from atleast one of the following residues of PCSK9: T468, R469, M470, A471,T472, R496, R499, E501, A502, Q503, R510, H512, F515, P540, P541, A542,E543, H565, W566, E567, V568, E569, R592, and E593 of SEQ ID NO: 3.

In some aspects, the invention comprises an isolated antigen bindingprotein. The antigen binding protein comprises: A) a CDRH1 of the CDRH1sequence in SEQ ID NO: 89, a CDRH2 of the CDRH2 sequence in SEQ ID NO:89, and a CDRH3 of the CDRH3 sequence in SEQ ID NO: 89, and B) a CDRL1of the CDRL1 sequence in SEQ ID NO:32, a CDRL2 of the CDRL2 sequence inSEQ ID NO:32, and a CDRL3 of the CDRL3 sequence in SEQ ID NO:32.

In some aspects, the invention comprises an isolated antigen bindingprotein that binds to a PCSK9 protein of SEQ ID NO: 1 where the bindingbetween said isolated antigen binding protein and a variant PCSK9protein is less than 50% of the binding between the isolated antigenbinding protein and the PCSK9 protein of SEQ ID NO: 1 and/or SEQ ID NO:303. In some embodiments, the variant PCSK9 protein comprises at leastone mutation of a residue at a position selected from the groupconsisting or comprising 207, 208, 185, 181, 439, 513, 538, 539, 132,351, 390, 413, 582, 162, 164, 167, 123, 129, 311, 313, 337, 519, 521,and 554, as shown in SEQ ID NO: 1. In some embodiments, the at least onemutation selected from the group comprising or consisting of R207E,D208R, E181R, R185E, R439E, E513R, V538R, E539R, T132R, S351R, A390R,A413R, and E582R. In some embodiments, the at least one mutation isselected from the group consisting of D162R, R164E, E167R, S123R, E129R,A311R, D313R, D337R, R519E, H521R, and Q554R.

In some aspects, the invention comprises an antigen binding protein thatbinds to a PCSK-9 protein of SEQ ID NO: 303 in a first manner and bindsto a variant of PCSK9 in a second manner. The PCSK9 variant has at leastone point mutation at a position selected from the group comprising orconsisting of: 207, 208, 185, 181, 439, 513, 538, 539, 132, 351, 390,413, 582, 162, 164, 167, 123, 129, 311, 313, 337, 519, 521, and 554 ofSEQ ID NO: 303 and/or SEQ ID NO: 1. In some embodiments, the firstmanner comprises a first EC50, a first Bmax, or a first EC50 and a firstBmax. In some embodiments, the second manner comprises a second EC50, asecond Bmax, or a second EC50 and a second Bmax. The value for the firstmanner is different from the value for the second manner. In someembodiments, the first manner comprises a first EC50, wherein the secondmanner involves a second EC50, and wherein the point mutation isselected from the group consisting or comprising: R207E, D208R, E181R,R185E, R439E, E513R, V538R, E539R, T132R, S351R, A390R, A413R, andE582R. In some embodiments, the first EC50 is at least 20% differentfrom the second EC50. In some embodiments, the first EC50 is at least50% different from the second EC50. In some embodiments, the second EC50is a larger numerical value than the first EC50. In some embodiments,the first EC50 is determined by a multiplex bead binding assay. In someembodiments, the second EC50 is greater than 1 um. In some embodiments,the antigen binding protein is a neutralizing antigen binding protein.In some embodiments, the neutralizing antigen binding protein is acompetitive neutralizing antigen binding protein. In some embodiments,the neutralizing antigen binding protein is a non-competitiveneutralizing antigen binding protein. In some embodiments, the firstmanner comprises a first Bmax and the second manner comprises a secondBmax that is different from the first Bmax. The PCSK9 variant has atleast one point mutation selected from the group consisting orcomprising: D162R, R164E, E167R, S123R, E129R, A311R, D313R, D337R,R519E, H521R, and Q554R. In some embodiments, the second Bmax is about10% of the first Bmax. In some embodiments, the first Bmax is at least20% different from the second Bmax. In some embodiments, the first Bmaxis at least 50% different from the second Bmax.

In some aspects, the invention comprises an isolated antigen bindingprotein that binds to a PCSK9 protein of SEQ ID NO: 3, wherein theepitope of the antigen binding protein includes at least one of thefollowing amino acids of SEQ ID NO: 1: 207, 208, 181, 185, 439, 513,538, 539, 132, 351, 390, 413, 582, 162, 164, 167, 123, 129, 311, 313,337, 519, 521, and 554.

In some aspects, the invention comprises an isolated neutralizingantigen binding protein that binds to a PCSK9 protein comprising theamino acid sequence of SEQ ID NO: 1, wherein the neutralizing antigenbinding protein decreases the LDLR lowering effect of PCSK9 on LDLR. Insome embodiments, the antigen binding protein is a LDLR non-competitiveneutralizing antigen binding protein. In some embodiments, the antigenbinding protein is a LDLR competitive neutralizing antigen bindingprotein.

In some aspects, the invention comprises an isolated antigen bindingprotein, wherein said antigen binding protein comprises: A) a CDRH1 ofthe CDRH1 sequence in SEQ ID NO: 49, a CDRH2 of the CDRH2 sequence inSEQ ID NO: 49, and a CDRH3 of the CDRH3 sequence in SEQ ID NO: 49, andB) a CDRL1 of the CDRL1 sequence in SEQ ID NO:23, a CDRL2 of the CDRL2sequence in SEQ ID NO:23, and a CDRL3 of the CDRL3 sequence in SEQ IDNO:23.

In some aspects, the invention comprises a composition comprising acrystallized PCSK9 protein and an antigen binding protein that binds toPCSK9. The composition comprises the crystallized PCSK9 protein is suchthat the three dimensional structure of the PCSK9 protein can bedetermined to a resolution of about 2.2 angstroms or better. In someembodiments, the antigen binding protein is an antibody or a fragmentthereof.

In some aspects, the invention comprises a crystallized PCSK9 proteinand at least an EGFa section of a LDLR protein, wherein the EGFa sectionof the LDLR protein is bound by a PCSK9 protein, wherein saidcrystallized PCSK9 protein is such that the three dimensional structureof the PCSK9 protein can be determined to a resolution of about 2.2angstroms or better. In some embodiments, the molecular model is on acomputer readable medium.

In some aspects, the invention comprises the use of an antigen bindingprotein as described herein, in the preparation of a medicament for thelowering of serum cholesterol.

In some aspects, the invention comprises the use of an antigen bindingprotein as described herein, in the preparation of a medicament fortreating or preventing a condition associated with elevated serumcholesterol levels in a subject.

In some aspects, the invention comprises an isolated antigen bindingprotein that binds PCSK9, the antigen binding protein comprising: A) aheavy chain complementary determining region (CDRH) selected from atleast one of the group consisting of: (i) a CDRH1 selected from theCDRH1 within the sequences selected from the group consisting of SEQ IDNOs: 67, 79, 89, and 49, (ii) a CDRH1 that differs in amino acidsequence from the CDRH1 of (i) by an amino acid addition, deletion orsubstitution of not more than two amino acids; and (iii) a CDRH1 aminoacid sequence selected from the group consisting ofX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀ (SEQ ID NO: 406), wherein X₁ is selected from thegroup consisting of G, X₂ is selected from the group consisting of Y, F,and G, X₃ is selected from the group consisting of T and S, X₄ isselected from the group consisting of L and F, X₅ is selected from thegroup consisting of T, S, and N, X₆ is selected from the groupconsisting of S and A, X₇ is selected from the group consisting of Y andF, X₈ is selected from the group consisting of G, S, and Y, X₉ isselected from the group consisting of I, M, and W, X₁₀ is selected fromthe group consisting of S, N and H, B) a light chain complementarydetermining region (CDRL) selected from at least one of the groupconsisting of: (i) a CDRL1 selected from the CDRL1 within the sequencesselected from the group consisting of SEQ ID NOs: 12, 32, 35, and 23,(ii) a CDRL1 that differs in amino acid sequence from the CDRL3 of (i)by an amino acid addition, deletion or substitution of not more than twoamino acids; and (iii) a CDRL1 amino acid sequence selected from thegroup consisting of X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄ (SEQ ID NO: 407),wherein X₁ is selected from the group consisting of T and no amino acid,X₂ is selected from the group consisting of G and S, X₃ is selected fromthe group consisting of S, T, and G, X₄ is selected from the groupconsisting of S, X₅ is selected from the group consisting of S, X₆ isselected from the group consisting of N, D, and S, X₇ is selected fromthe group consisting of I, V, and N, X₈ is selected from the groupconsisting of G and I, X₉ is selected from the group consisting of A andG, X₁₀ is selected from the group consisting of G, Y, S, and N, X₁₁ isselected from the group consisting of Y and N, X₁₂ is selected from thegroup consisting of D, S, T, and F, X₁₃ is selected from the groupconsisting of V, X₁₄ is selected from the group consisting of S, N, andH. One of skill in the art will appreciate that a single ABP or antibodycan meet one or more of the above options and still fall within thedescribed invention for this embodiment.

In some aspects, the invention comprises an isolated antigen bindingprotein that binds PCSK9, the antigen binding protein comprising: A) aheavy chain complementary determining region (CDRH) selected from atleast one of the group consisting of the following: (i) a CDRH2 selectedfrom the CDRH2 within the sequences selected from the group consistingof SEQ ID NOs: 67, 79, 89, and 49, (ii) a CDRH2 that differs in aminoacid sequence from the CDRH2 of (i) by an amino acid addition, deletionor substitution of not more than two amino acids; and (iii) a CDRH2amino acid sequence selected from the group consisting ofX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇ (SEQ ID NO: 408), wherein X₁is selected from the group consisting of W, S, L and no amino acid, X₂is selected from the group consisting of V, I, and E, X₃ is selectedfrom the group consisting of S, W, and I, X₄ is selected from the groupconsisting of F, S, and N, X₅ is selected from the group consisting ofY, S, D, and H, X₆ is selected from the group consisting of N, S, and G,X₇ is selected from the group consisting of S and G, X₈ is selected fromthe group consisting of N, Y, D, and R, X₉ is selected from the groupconsisting of T, I, and E, X₁₀ is selected from the group consisting ofN, S, Y, and D, X₁₁ is selected from the group consisting of Y, X₁₂ isselected from the group consisting of A and N, X₁₃ is selected from thegroup consisting of Q, D, and P, X₁₄ is selected from the groupconsisting of K and S, X₁₅ is selected from the group consisting of L,and V, X₁₆ is selected from the group consisting of Q and K, X₁₇ isselected from the group consisting of G and S, B) a light chaincomplementary determining region (CDRL) selected from at least one ofthe group consisting of the following: (i) a CDRL2 selected from theCDRL3 within the sequences selected from the group consisting of SEQ IDNOs: 12, 32, 35, and 23, (ii) a CDRL2 that differs in amino acidsequence from the CDRL3 of (i) by an amino acid addition, deletion orsubstitution of not more than two amino acids; and (iii) a CDRL2 aminoacid sequence selected from the group consisting of X₁X₂X₃X₄X₅X₆X₇ (SEQID NO: 409), wherein X₁ is selected from the group consisting of G, E,S, and D, X₂ is selected from the group consisting of N, V, and Y, X₃ isselected from the group consisting of S and N, X₄ is selected from thegroup consisting of N, Q, and K, X₅ is selected from the groupconsisting of R, X₆ is selected from the group consisting of P, X₇ isselected from the group consisting of S.

In some aspects, the invention comprises An isolated antigen bindingprotein that binds PCSK9, the antigen binding protein comprising: A) aheavy chain complementary determining region (CDRH) selected from atleast one of the group consisting of the following: (i) a CDRH3 selectedfrom the CDRH3 within the sequences selected from the group consistingof SEQ ID NOs: 67, 79, 89, and 49, (ii) a CDRH3 that differs in aminoacid sequence from the CDRH3 of (i) by an amino acid addition, deletionor substitution of not more than two amino acids; and (iii) a CDRH3amino acid sequence selected from the group consisting ofX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄ (SEQ ID NO: 410), wherein X₁ isselected from the group consisting of D, and no amino acid, X₂ isselected from the group consisting of Y, A, and no amino acid, X₃ isselected from the group consisting of D, I, and no amino acid, X₄ isselected from the group consisting of F, A, and no amino acid, X₅ isselected from the group consisting of W, A, and no amino acid, X₆ isselected from the group consisting of S, L, and no amino acid, X₇ isselected from the group consisting of A, Y, G, and no amino acid, X₈ isselected from the group consisting of Y, Q, and no amino acid, X₉ isselected from the group consisting of G, Y, and L, X₁₀ is selected fromthe group consisting of Y, D, and V, X₁₁ is selected from the groupconsisting of G, A, and P, X₁₂ is selected from the group consisting ofM and F, X₁₃ is selected from the group consisting of D, X₁₄ is selectedfrom the group consisting of V and Y, and B) a light chain complementarydetermining region (CDRL) selected from at least one of the groupconsisting of the following: (i) a CDRL3 selected from the CDRL3 withinthe sequences selected from the group consisting of SEQ ID NOs: 12, 32,35, and 23, (ii) a CDRL3 that differs in amino acid sequence from theCDRL3 of (i) by an amino acid addition, deletion or substitution of notmore than two amino acids; and (iii) a CDRL3 amino acid sequenceselected from the group consisting of X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁ (SEQ IDNO: 411), wherein X₁ is selected from the group consisting of Q, A, G,and no amino acid, X₂ is selected from the group consisting of S, V, T,and no amino acid, X₃ is selected from the group consisting of Y, N, andW, X₄ is selected from the group consisting of S and D, X₅ is selectedfrom the group consisting of S, Y, and D, X₆ is selected from the groupconsisting of S and T, X₇ is selected from the group consisting of L andS, X₈ is selected from the group consisting of S, T, and N, X₉ isselected from the group consisting of G, S, and A, X₁₀ is selected fromthe group consisting of S, M, W, and Y, and X₁₁ is selected from thegroup consisting of V. In some embodiments, any of the above amino acidscan be replaced by a conservative amino acid substitution.

In some aspects, the invention comprises an isolated antigen bindingprotein that binds PCSK9, the antigen binding protein comprises A) aheavy chain complementary determining region (CDRH) selected from atleast one of the group consisting of (i) a CDRH1 selected from the CDRH1within the sequences selected from the group consisting of SEQ ID NOs:47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, and 58, (ii) a CDRH1 thatdiffers in amino acid sequence from the CDRH1 of (i) by an amino acidaddition, deletion or substitution of not more than two amino acids; and(iii) a CDRH1 amino acid sequence selected from the group consisting ofX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀ (SEQ ID NO: 412), wherein X₁ is selected from thegroup consisting of G, P, and A, X₂ is selected from the groupconsisting of Y, W, F, T, and S, X₃ is selected from the groupconsisting of T, P, S and A, C, V, L, and I, X₄ is selected from thegroup consisting of L, F, I, V, M, A, and Y, X₅ is selected from thegroup consisting of T, P, S, and A, X₆ is selected from the groupconsisting of S, T, A, and C, X₇ is selected from the group consistingof Y, W, F, T, and S, X₈ is selected from the group consisting of G, P,and A, X₉ is selected from the group consisting of I, L, V, M, A, and F,X₁₀ is selected from the group consisting of S, T, A, and C, B) a lightchain complementary determining region (CDRL) selected from at least oneof the group consisting of: (i) a CDRL1 selected from the CDRL1 withinthe sequences selected from the group consisting of SEQ ID NOs: 14, 15,16, 17, 18, 19, 20, 21, 22, 23, and 24, (ii) a CDRL1 that differs inamino acid sequence from the CDRL3 of (i) by an amino acid addition,deletion or substitution of not more than two amino acids; and (iii) aCDRL1 amino acid sequence selected from the group consisting ofX₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄ (SEQ ID NO: 413), wherein, X₁ isselected from the group consisting of T and S, X₂ is selected from thegroup consisting of G, P, and A, X₃ is selected from the groupconsisting of T, and S, X₄ is selected from the group consisting of S N,T, A, C, and Q, X₅ is selected from the group consisting of S, T, A, andC, X₆ is selected from the group consisting of D, and E, X₇ is selectedfrom the group consisting of V, I, M, L, F, and A, X₈ is selected fromthe group consisting of G, P, and A, X₉ is selected from the groupconsisting of G, A, R, P, V, L, I, K, Q, and N, X₁₀ is selected from thegroup consisting of Y, W, F, T, and S, X₁₁ is selected from the groupconsisting of N, and Q, X₁₂ is selected from the group consisting of Y,S, W, F, T, A, and C, X₁₃ is selected from the group consisting of V, I,M, L, F, and A, X₁₄ is selected from the group consisting of S, T, A,and C.

In some aspects, the invention comprises an isolated antigen bindingprotein that binds PCSK9, the antigen binding protein comprising: A) aheavy chain complementary determining region (CDRH) selected from atleast one of the group consisting of: (i) a CDRH2 selected from theCDRH2 within the sequences selected from the group consisting of SEQ IDNOs: 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, and 58, (ii) a CDRH2that differs in amino acid sequence from the CDRH2 of (i) by an aminoacid addition, deletion or substitution of not more than two aminoacids; and (iii) a CDRH2 amino acid sequence selected from the groupconsisting of X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁X₁₂X₁₃X₁₄X₁₅X₁₆X₁₇, (SEQ ID NO:414), wherein X₁ is selected from the group consisting of W, Y, and F,X₂ is selected from the group consisting of V, I, M, L, F, and A, X₃ isselected from the group consisting of S, T, A, and C, X₄ is selectedfrom the group consisting of A, F, V, L, I, Y, and M, X₅ is selectedfrom the group consisting of Y, W, F, T, and S, X₆ is selected from thegroup consisting of N and Q, X₇ is selected from the group consisting ofG, P, and A, X₈ is selected from the group consisting of N, and Q, X₉ isselected from the group consisting of T, and S, X₁₀ is selected from thegroup consisting of N, and Q, X₁₁ is selected from the group consistingof Y, W, F, T, and S, X₁₂ is selected from the group consisting of A, V,L, and I, X₁₃ is selected from the group consisting of Q, E, N, and D,X₁₄ is selected from the group consisting of K, R, Q, and N, X₁₅ isselected from the group consisting of L, F, V, I, M, A, and Y, X₁₆ isselected from the group consisting of Q, and N, X₁₇ is selected from thegroup consisting of G, P, and A, B) a light chain complementarydetermining region (CDRL) selected from at least one of the groupconsisting of: (i) a CDRL2 selected from the CDRL3 within the sequencesselected from the group consisting of SEQ ID NOs: 14, 15, 16, 17, 18,19, 20, 21, 22, 23, and 24, (ii) a CDRL2 that differs in amino acidsequence from the CDRL3 of (i) by an amino acid addition, deletion orsubstitution of not more than two amino acids; and (iii) a CDRL2 aminoacid sequence selected from the group consisting of X₁X₂X₃X₄X₅X₆X₇ (SEQID NO: 415), wherein X₁ is selected from the group consisting of E, andD, X₂ is selected from the group consisting of V, I, M, L, F, and A, X₃is selected from the group consisting of S, T, A, and C, X₄ is selectedfrom the group consisting of N, and Q, X₅ is selected from the groupconsisting of R, K, Q, and N, X₆ is selected from the group consistingof P, and A, X₇ is selected from the group consisting of S, T, A, and C.

In some aspects, the invention comprises an isolated antigen bindingprotein that binds PCSK9, the antigen binding protein comprising: A) aheavy chain complementary determining region (CDRH) selected from atleast one of the group consisting of (i) a CDRH3 selected from the CDRH3within the sequences selected from the group consisting of SEQ ID NOs:47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, and 58, (ii) a CDRH3 thatdiffers in amino acid sequence from the CDRH3 of (i) by an amino acidaddition, deletion or substitution of not more than two amino acids; and(iii) a CDRH3 amino acid sequence selected from the group consisting ofX₁X₂X₃X₄X₅X₆ (SEQ ID NO: 416), wherein X₁ is selected from the groupconsisting of G, P, A and no amino acid, X₂ is selected from the groupconsisting of Y, W, F, T, and S, X₃ is selected from the groupconsisting of G, V, P, A, I, M, L, and F, X₄ is selected from the groupconsisting of M, L, F, and I, X₅ is selected from the group consistingof D, and E, X₆ is selected from the group consisting of V, I, M, L, F,and A, B) a light chain complementary determining region (CDRL) selectedfrom at least one of the group consisting of: (i) a CDRL3 selected fromthe CDRL3 within the sequences selected from the group consisting of SEQID NOs: 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24, (ii) a CDRL3that differs in amino acid sequence from the CDRL3 of (i) by an aminoacid addition, deletion or substitution of not more than two aminoacids; and (iii) a CDRL3 amino acid sequence selected from the groupconsisting of X₁X₂X₃X₄X₅X₆X₇X₈X₉ (SEQ ID NO: 417), wherein X₁ isselected from the group consisting of S, N, T, A, C, and Q, X₂ isselected from the group consisting of S, T, A, and C, X₃ is selectedfrom the group consisting of Y, W, F, T, and S, X₄ is selected from thegroup consisting of T, and S, X₅ is selected from the group consistingof S, T, A, and C, X₆ is selected from the group consisting of S, T, A,and C, X₇ is selected from the group consisting of N, S, Q, T, A, and C,X₈ is selected from the group consisting of M, V, L, F, I, and A, X₉ isselected from the group consisting of V, I, M, L, F, and A.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A depicts an amino acid sequence of the mature form of the PCSK9with the pro-domain underlined.

FIGS. 1B ₁-1B₄ depict amino acid and nucleic acid sequences of PCSK9with the pro-domain underlined and the signal sequence in bold.

FIGS. 2A-2D are sequence comparison tables of various light chains ofvarious antigen binding proteins. FIG. 2C continues the sequence startedin FIG. 2A. FIG. 2D continues the sequence started on FIG. 2B.

FIGS. 3A-3D are sequence comparison tables of various heavy chains ofvarious antigen binding proteins. FIG. 3C continues the sequence startedin FIG. 3A. FIG. 3D continues the sequence started on FIG. 3B.

FIGS. 3E-3JJ depict the amino acid and nucleic acid sequences for thevariable domains of some embodiments of the antigen binding proteins.

FIG. 3KK depicts the amino acid sequences for various constant domains.

FIGS. 3LL-3BBB depict the amino acid and nucleic acid sequences for thevariable domains of some embodiments of the antigen binding proteins.

FIGS. 3CCC-3JJJ are sequence comparison tables of various heavy andlight chains of some embodiments of the antigen binding proteins.

FIG. 4A is a binding curve of an antigen binding protein to human PCSK9.

FIG. 4B is a binding curve of an antigen binding protein to human PCSK9.

FIG. 4C is a binding curve of an antigen binding protein to cynomolgusPCSK9.

FIG. 4D is a binding curve of an antigen binding protein to cynomolgusPCSK9.

FIG. 4E is a binding curve of an antigen binding protein to mouse PCSK9.

FIG. 4F is a binding curve of an antigen binding protein to mouse PCSK9.

FIG. 5A depicts the results of an SDS PAGE experiment involving PCSK9and various antigen binding proteins demonstrating the relative purityand concentration of the proteins.

FIGS. 5B and 5C depict graphs from biacore solution equilibrium assaysfor 21B12.

FIG. 5D depicts the graph of the kinetics from a biacore capture assay.

FIG. 5E depicts a bar graph depicting binning results for three ABPs.

FIG. 6A is an inhibition curve of antigen binding protein 31H4 IgG2 toPCSK9 in an in vitro PCSK9:LDLR binding assay

FIG. 6B is an inhibition curve of antigen binding protein 31H4 IgG4 toPCSK9 in an in vitro PCSK9:LDLR binding assay.

FIG. 6C is an inhibition curve of antigen binding protein 21B12 IgG2 toPCSK9 in an in vitro PCSK9:LDLR binding assay.

FIG. 6D is an inhibition curve of antigen binding protein 21B12 IgG4 toPCSK9 in an in vitro PCSK9:LDLR binding assay.

FIG. 7A is an inhibition curve of antigen binding protein 31H4 IgG2 inthe cell LDL uptake assay showing the effect of the ABP to reduce theLDL uptake blocking effects of PCSK9

FIG. 7B is an inhibition curve of antigen binding protein 31H4 IgG4 inthe cell LDL uptake assay showing the effect of the ABP to reduce theLDL uptake blocking effects of PCSK9

FIG. 7C is an inhibition curve of antigen binding protein 21B12 IgG2 inthe cell LDL uptake assay showing the effect of the ABP to reduce theLDL uptake blocking effects of PCSK9

FIG. 7D is an inhibition curve of antigen binding protein 21B12 IgG4 inthe cell LDL uptake assay showing the effect of the ABP to reduce theLDL uptake blocking effects of PCSK9

FIG. 8A is a graph depicting the serum cholesterol lowering ability inmice of ABP 31H4, changes relative to the IgG control treated mice (*p<0.01).

FIG. 8B is a graph depicting the serum cholesterol lowering ability inmice of ABP 31H4, changes relative to time=zero hours (# p, 0.05).

FIG. 8C is a graph depicting the effect of ABP 31H4 on HDL cholesterollevels in C57B1/6 mice (* p<0.01).

FIG. 8D is a graph depicting the effect of ABP 31H4 on HDL cholesterollevels in C57B1/6 mice (# p<0.05).

FIG. 9 depicts a western blot analysis of the ability of ABP 31H4 toenhance the amount of liver LDLR protein present after various timepoints.

FIG. 10A is a graph depicting the ability of an antigen binding protein31H4 to lower total serum cholesterol in wild type mice, relative.

FIG. 10B is a graph depicting the ability of an antigen binding protein31H4 to lower HDL in wild type mice.

FIG. 10C is a graph depicting the serum cholesterol lowering ability ofvarious antigen binding proteins 31H4 and 16F12.

FIG. 11A depicts an injection protocol for testing the duration andability of antigen binding proteins to lower serum cholesterol.

FIG. 11B is a graph depicting the results of the protocol in FIG. 11A.

FIG. 12A depicts LDLR levels in response to the combination of a statinand ABP 21B12 in HepG2 cells.

FIG. 12B depicts LDLR levels in response to the combination of a statinand ABP 31H4 in HepG2 cells.

FIG. 12C depicts LDLR levels in response to the combination of a statinand ABP 25A7.1, a normeutralizing antibody, (in contrast the “25A7” aneutralizing antibody) in HepG2 cells.

FIG. 12D depicts LDLR levels in response to the combination of a statinand ABP 21B12 in HepG2 cells overexpressing PCSK9.

FIG. 12E depicts LDLR levels in response to the combination of a statinand ABP 31H4 in HepG2 cells overexpressing PCSK9.

FIG. 12F depicts LDLR levels in response to the combination of a statinand ABP 25A7.1, a normeutralizing antibody, (in contrast the “25A7” aneutralizing antibody) in HepG2 cells overexpressing PCSK9.

FIG. 13A depicts the various light chain amino acid sequences of variousABPs to PCSK9. The dots (.) indicate no amino acid.

FIG. 13B depicts a light chain cladogram for various ABPs to PCSK9.

FIG. 13C depicts the various heavy chain amino acid sequences of variousABPs to PCSK9. The dots (.) indicate no amino acid.

FIG. 13D depicts a heavy chain dendrogram for various ABPs to PCSK9.

FIG. 13E depicts a comparison of light and heavy CDRs and designation ofgroups from which to derive consensus.

FIG. 13F depicts the consensus sequences for Groups 1 and 2.

FIG. 13G depicts the consensus sequences for Groups 3 and 4.

FIG. 13H depicts the consensus sequences for Groups 1 and 2. The dots(.) indicated identical residues.

FIG. 13I depicts the consensus sequences for Group 2. The dots (.)indicated identical residues.

FIG. 13J depicts the consensus sequences for Groups 3 and 4. The dots(.) indicated identical residues.

FIG. 14A is a graph depicting in vivo LDL lowering ability of variousABPs (at 10 mg/kg).

FIG. 14B is a graph depicting in vivo LDL lowering ability of variousABPs (at 30 mg/kg).

FIG. 15A and FIG. 15B are sequence comparison tables of various lightchains of various embodiments of antigen binding proteins. FIG. 15Bcontinues the sequence started in FIG. 15A.

FIG. 15C and FIG. 15D are sequence comparison tables of various lightchains of various embodiments of antigen binding proteins. FIG. 15Dcontinues the sequence started in FIG. 15C.

FIG. 16A is a depiction of a gel used to test the ability of Ab 21B12 tobind to the ProCat or VD sections of PCSK9.

FIG. 16B is a depiction of a gel used to test the ability of Ab 31H4 tobind to the ProCat or VD sections of PCSK9.

FIG. 17 is a depiction of the structure of PCSK9 and the EGFa section ofLDLR.

FIG. 18A is a depiction of the structure of PCSK9 and the 31H4 Ab.

FIG. 18B is a depiction of the structure of PCSK9 and the 31H4 Ab.

FIG. 19A is a depiction of the structure of PCSK9, the 31H4 Ab, and the21B12 Ab.

FIG. 19B is a depiction of the structure of PCSK9 and the 21B12 Ab.

FIG. 20A is a depiction of the structure of PCSK9 and EGFa from the LDLRsuperimposed with the structure of antibodies 31H4 and 21B12 bound toPCSK9.

FIG. 20B is a depiction of the structural model of PCSK9 and LDLR.

FIG. 20C is a depiction of the structural model of PCSK9 and LDLR froman alternative perspective.

FIG. 20D is a depiction of the structural model of PCSK9 and LDLR withstructural representations of 31H4 and 21B12 included.

FIG. 20E is a depiction of the structural model in FIG. 20D, rotated 90degrees about the noted axis.

FIG. 20F is a depiction of the structural model in FIG. 20D rotated 180degrees about the noted axis.

FIG. 21A is a depiction of the structure of PCSK9 and 31A4.

FIG. 21B is a depiction of the structure of PCSK9 and 31A4.

FIG. 21C is a depiction of the structure of PCSK9 and 31A4.

FIG. 21D is a depiction of the structural model of full length PCSK9 and31A4.

FIG. 22 is a set of ABP sequences identifying various differencesbetween the human ABP sequences and the ABP sequences that were raisedin E. coli and used for the crystal structures.

FIG. 23 is a table depicting the various binning results.

FIG. 23A is a first part of a table depicting the various binningresults.

FIG. 23B is a second part of a table depicting the various binningresults.

FIG. 23C is a third part of a table depicting the various binningresults.

FIG. 23D is a fourth part of a table depicting the various binningresults.

FIG. 24A is a depiction of a western blot under non-reduced conditions.

FIG. 24B is a depiction of a western blot under reduced conditions.

FIG. 25A is a depiction of the surface coverage of PCSK9.

FIG. 25B is a depiction of the surface coverage of PCSK9.

FIG. 25C is a depiction of the surface coverage of PCSK9.

FIG. 25D is a depiction of the surface coverage of PCSK9.

FIG. 25E is a depiction of the surface coverage of PCSK9.

FIG. 25F is a depiction of the surface coverage of PCSK9.

FIG. 26 is a sequence comparison of the PCSK9 amino acid sequence andall of the residues that were mutated in PCSK9 variants to examine theepitopes of the various antibodies.

FIG. 27A depicts the 21B12 epitope hits, as mapped onto a crystalstructure of PCSK9 with the 21B12.

FIG. 27B depicts the 31H4 epitope hits, as mapped onto a crystalstructure of PCSK9 with 31H4 and 21B1.

FIG. 27C depicts the 31A4 epitope hits, as mapped onto a crystalstructure of PCSK9 with 31H4 and 21B12.

FIG. 27D depicts the 12H11 epitope hits, as mapped onto the crystalstructure of PCSK9 with 31H4 and 21B12.

FIG. 27E depicts the 3C4 epitope hits, as mapped onto the crystalstructure of PCSK9 with 31H4 and 21B12.

FIG. 28A is a graph demonstrating the binding ability of the variousABPs to various parts of PCSK9.

FIG. 28B is a graph demonstrating the binding ability of the variousABPs to various parts of PCSK9.

FIG. 28C is a graph comparing the LDLR binding ability of two ABPs.

FIG. 28D is a graph comparing the cell LDL uptake activity of two ABPs.

DETAILED DESCRIPTION OF CERTAIN EXEMPLARY EMBODIMENTS

Antigen binding proteins (such as antibodies and functional bindingfragments thereof) that bind to PCSK9 are disclosed herein. In someembodiments, the antigen binding proteins bind to PCSK9 and preventPCSK9 from functioning in various ways. In some embodiments, the antigenbinding proteins block or reduce the ability of PCSK9 to interact withother substances. For example, in some embodiments, the antigen bindingprotein binds to PCSK9 in a manner that prevents or reduces thelikelihood that PCSK9 will bind to LDLR. In other embodiments, antigenbinding proteins bind to PCSK9 but do not block PCSK9's ability tointeract with LDLR. In some embodiments, the antigen binding proteinsare human monoclonal antibodies.

As will be appreciated by one of skill in the art, in light of thepresent disclosure, altering the interactions between PCSK9 and LDLR canincrease the amount of LDLR available for binding to LDL, which in turndecreases the amount of serum LDL in a subject, resulting in a reductionin the subject's serum cholesterol level. As such, antigen bindingproteins to PCSK9 can be used in various methods and compositions fortreating subjects with elevated serum cholesterol levels, at risk ofelevated serum cholesterol levels, or which could benefit from areduction in their serum cholesterol levels. Thus, various methods andtechniques for lowering, maintaining, or preventing an increase in serumcholesterol are also described herein. In some embodiments, the antigenbinding protein allows for binding between PCSK9 and LDLR, but theantigen binding protein prevents or reduces the adverse activity ofPCSK9 on LDLR. In some embodiments, the antigen binding protein preventsor reduces the binding of PCSK9 to LDLR.

For convenience, the following sections generally outline the variousmeanings of the terms used herein. Following this discussion, generalaspects regarding antigen binding proteins are discussed, followed byspecific examples demonstrating the properties of various embodiments ofthe antigen binding proteins and how they can be employed.

DEFINITIONS AND EMBODIMENTS

It is to be understood that both the foregoing general description andthe following detailed description are exemplary and explanatory onlyand are not restrictive of the invention as claimed. In thisapplication, the use of the singular includes the plural unlessspecifically stated otherwise. In this application, the use of “or”means “and/or” unless stated otherwise. Furthermore, the use of the term“including”, as well as other forms, such as “includes” and “included”,is not limiting. Also, terms such as “element” or “component” encompassboth elements and components comprising one unit and elements andcomponents that comprise more than one subunit unless specificallystated otherwise. Also, the use of the term “portion” can include partof a moiety or the entire moiety.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.All documents, or portions of documents, cited in this application,including but not limited to patents, patent applications, articles,books, and treatises, are hereby expressly incorporated by reference intheir entirety for any purpose. As utilized in accordance with thepresent disclosure, the following terms, unless otherwise indicated,shall be understood to have the following meanings:

The term “proprotein convertase subtilisin kexin type 9” or “PCSK9”refers to a polypeptide as set forth in SEQ ID NO: 1 and/or 3 orfragments thereof, as well as related polypeptides, which include, butare not limited to, allelic variants, splice variants, derivativevariants, substitution variants, deletion variants, and/or insertionvariants including the addition of an N-terminal methionine, fusionpolypeptides, and interspecies homologs. In certain embodiments, a PCSK9polypeptide includes terminal residues, such as, but not limited to,leader sequence residues, targeting residues, amino terminal methionineresidues, lysine residues, tag residues and/or fusion protein residues.“PCSK9” has also been referred to as FH3, NARC1, HCHOLA3, proproteinconvertase subtilisin/kexin type 9, and neural apoptosis regulatedconvertase 1. The PCSK9 gene encodes a proprotein convertase proteinthat belongs to the proteinase K subfamily of the secretory subtilasefamily. The term “PCSK9” denotes both the proprotein and the productgenerated following autocatalysis of the proprotein. When only theautocatalyzed product is being referred to (such as for an antigenbinding protein that selectively binds to the cleaved PCSK9), theprotein can be referred to as the “mature,” “cleaved”, “processed” or“active” PCSK9. When only the inactive form is being referred to, theprotein can be referred to as the “inactive”, “pro-form”, or“unprocessed” form of PCSK9. The term PCSK9 as used herein also includesnaturally occurring alleles, such as the mutations D374Y, S127R andF216L. The term PCSK9 also encompasses PCSK9 molecules incorporatingpost-translational modifications of the PCSK9 amino acid sequence, suchas PCSK9 sequences that have been glycosylated, PEGylated, PCSK9sequences from which its signal sequence has been cleaved, PCSK9sequence from which its pro domain has been cleaved from the catalyticdomain but not separated from the catalytic domain (e.g., FIGS. 1A and1B).

The term “PCSK9 activity” includes any biological effect of PCSK9. Incertain embodiments, PCSK9 activity includes the ability of PCSK9 tointeract or bind to a substrate or receptor. In some embodiments, PCSK9activity is represented by the ability of PCSK9 to bind to a LDLreceptor (LDLR). In some embodiments, PCSK9 binds to and catalyzes areaction involving LDLR. In some embodiments, PCSK9 activity includesthe ability of PCSK9 to alter (e.g., reduce) the availability of LDLR.In some embodiments, PCSK9 activity includes the ability of PCSK9 toincrease the amount of LDL in a subject. In some embodiments, PCSK9activity includes the ability of PCSK9 to decrease the amount of LDLRthat is available to bind to LDL. In some embodiments, “PCSK9 activity”includes any biological activity resulting from PCSK9 signaling.Exemplary activities include, but are not limited to, PCSK9 binding toLDLR, PCSK9 enzyme activity that cleaves LDLR or other proteins, PCSK9binding to proteins other than LDLR that facilitate PCSK9 action, PCSK9altering APOB secretion (Sun X-M et al, “Evidence for effect of mutantPCSK9 on apoliprotein B secretion as the cause of unusually severedominant hypercholesterolemia, Human Molecular Genetics 14: 1161-1169,2005 and Ouguerram K et al, “Apolipoprotein B100 metabolism inautosomal-dominant hypercholesterolemia related to mutations in PCSK9,Arterioscler thromb Vasc Biol. 24: 1448-1453, 2004), PCSK9's role inliver regeneration and neuronal cell differentiation (Seidah N G et al,“The secretory proprotein convertase neural apoptosis-regulatedconvertase 1 (NARC-1): Liver regeneration and neuronal differentiation”PNAS 100: 928-933, 2003), and PCSK9s role in hepatic glucose metabolism(Costet et al., “Hepatic PCSK9 expression is regulated by nutritionalstatus via insulin and sterol regulatory element-binding protein 1c” J.Biol. Chem. 281(10):6211-18, 2006).

The term “hypercholesterolemia,” as used herein, refers to a conditionin which cholesterol levels are elevated above a desired level. In someembodiments, this denotes that serum cholesterol levels are elevated. Insome embodiments, the desired level takes into account various “riskfactors” that are known to one of skill in the art (and are described orreferenced herein).

The term “polynucleotide” or “nucleic acid” includes bothsingle-stranded and double-stranded nucleotide polymers. The nucleotidescomprising the polynucleotide can be ribonucleotides ordeoxyribonucleotides or a modified form of either type of nucleotide.Said modifications include base modifications such as bromouridine andinosine derivatives, ribose modifications such as 2′,3′-dideoxyribose,and internucleotide linkage modifications such as phosphorothioate,phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,phosphoroanilothioate, phoshoraniladate and phosphoroamidate.

The term “oligonucleotide” means a polynucleotide comprising 200 orfewer nucleotides. In some embodiments, oligonucleotides are 10 to 60bases in length. In other embodiments, oligonucleotides are 12, 13, 14,15, 16, 17, 18, 19, or 20 to 40 nucleotides in length. Oligonucleotidescan be single stranded or double stranded, e.g., for use in theconstruction of a mutant gene. Oligonucleotides can be sense orantisense oligonucleotides. An oligonucleotide can include a label,including a radiolabel, a fluorescent label, a hapten or an antigeniclabel, for detection assays. Oligonucleotides can be used, for example,as PCR primers, cloning primers or hybridization probes.

An “isolated nucleic acid molecule” means a DNA or RNA of genomic, mRNA,cDNA, or synthetic origin or some combination thereof which is notassociated with all or a portion of a polynucleotide in which theisolated polynucleotide is found in nature, or is linked to apolynucleotide to which it is not linked in nature. For purposes of thisdisclosure, it should be understood that “a nucleic acid moleculecomprising” a particular nucleotide sequence does not encompass intactchromosomes. Isolated nucleic acid molecules “comprising” specifiednucleic acid sequences can include, in addition to the specifiedsequences, coding sequences for up to ten or even up to twenty otherproteins or portions thereof, or can include operably linked regulatorysequences that control expression of the coding region of the recitednucleic acid sequences, and/or can include vector sequences.

Unless specified otherwise, the left-hand end of any single-strandedpolynucleotide sequence discussed herein is the 5′ end; the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction; sequenceregions on the DNA strand having the same sequence as the RNA transcriptthat are 5′ to the 5′ end of the RNA transcript are referred to as“upstream sequences;” sequence regions on the DNA strand having the samesequence as the RNA transcript that are 3′ to the 3′ end of the RNAtranscript are referred to as “downstream sequences.”

The term “control sequence” refers to a polynucleotide sequence that canaffect the expression and processing of coding sequences to which it isligated. The nature of such control sequences can depend upon the hostorganism. In particular embodiments, control sequences for prokaryotescan include a promoter, a ribosomal binding site, and a transcriptiontermination sequence. For example, control sequences for eukaryotes caninclude promoters comprising one or a plurality of recognition sites fortranscription factors, transcription enhancer sequences, andtranscription termination sequence. “Control sequences” can includeleader sequences and/or fusion partner sequences.

The term “vector” means any molecule or entity (e.g., nucleic acid,plasmid, bacteriophage or virus) used to transfer protein codinginformation into a host cell.

The term “expression vector” or “expression construct” refers to avector that is suitable for transformation of a host cell and containsnucleic acid sequences that direct and/or control (in conjunction withthe host cell) expression of one or more heterologous coding regionsoperatively linked thereto. An expression construct can include, but isnot limited to, sequences that affect or control transcription,translation, and, if introns are present, affect RNA splicing of acoding region operably linked thereto.

As used herein, “operably linked” means that the components to which theterm is applied are in a relationship that allows them to carry outtheir inherent functions under suitable conditions. For example, acontrol sequence in a vector that is “operably linked” to a proteincoding sequence is ligated thereto so that expression of the proteincoding sequence is achieved under conditions compatible with thetranscriptional activity of the control sequences.

The term “host cell” means a cell that has been transformed, or iscapable of being transformed, with a nucleic acid sequence and therebyexpresses a gene of interest. The term includes the progeny of theparent cell, whether or not the progeny is identical in morphology or ingenetic make-up to the original parent cell, so long as the gene ofinterest is present.

The term “transfection” means the uptake of foreign or exogenous DNA bya cell, and a cell has been “transfected” when the exogenous DNA hasbeen introduced inside the cell membrane. A number of transfectiontechniques are well known in the art and are disclosed herein. See,e.g., Graham et al., 1973, Virology 52:456; Sambrook et al., 2001,Molecular Cloning: A Laboratory Manual, supra; Davis et al., 1986, BasicMethods in Molecular Biology, Elsevier; Chu et al., 1981, Gene 13:197.Such techniques can be used to introduce one or more exogenous DNAmoieties into suitable host cells.

The term “transformation” refers to a change in a cell's geneticcharacteristics, and a cell has been transformed when it has beenmodified to contain new DNA or RNA. For example, a cell is transformedwhere it is genetically modified from its native state by introducingnew genetic material via transfection, transduction, or othertechniques. Following transfection or transduction, the transforming DNAcan recombine with that of the cell by physically integrating into achromosome of the cell, or can be maintained transiently as an episomalelement without being replicated, or can replicate independently as aplasmid. A cell is considered to have been “stably transformed” when thetransforming DNA is replicated with the division of the cell.

The terms “polypeptide” or “protein” means a macromolecule having theamino acid sequence of a native protein, that is, a protein produced bya naturally-occurring and non-recombinant cell; or it is produced by agenetically-engineered or recombinant cell, and comprise moleculeshaving the amino acid sequence of the native protein, or moleculeshaving deletions from, additions to, and/or substitutions of one or moreamino acids of the native sequence. The term also includes amino acidpolymers in which one or more amino acids are chemical analogs of acorresponding naturally-occurring amino acid and polymers. The terms“polypeptide” and “protein” specifically encompass PCSK9 antigen bindingproteins, antibodies, or sequences that have deletions from, additionsto, and/or substitutions of one or more amino acid of antigen-bindingprotein. The term “polypeptide fragment” refers to a polypeptide thathas an amino-terminal deletion, a carboxyl-terminal deletion, and/or aninternal deletion as compared with the full-length native protein. Suchfragments can also contain modified amino acids as compared with thenative protein. In certain embodiments, fragments are about five to 500amino acids long. For example, fragments can be at least 5, 6, 8, 10,14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 aminoacids long. Useful polypeptide fragments include immunologicallyfunctional fragments of antibodies, including binding domains. In thecase of a PCSK9-binding antibody, useful fragments include but are notlimited to a CDR region, a variable domain of a heavy and/or lightchain, a portion of an antibody chain or just its variable regionincluding two CDRs, and the like.

The term “isolated protein” referred means that a subject protein (1) isfree of at least some other proteins with which it would normally befound, (2) is essentially free of other proteins from the same source,e.g., from the same species, (3) is expressed by a cell from a differentspecies, (4) has been separated from at least about 50 percent ofpolynucleotides, lipids, carbohydrates, or other materials with which itis associated in nature, (5) is operably associated (by covalent ornoncovalent interaction) with a polypeptide with which it is notassociated in nature, or (6) does not occur in nature. Typically, an“isolated protein” constitutes at least about 5%, at least about 10%, atleast about 25%, or at least about 50% of a given sample. Genomic DNA,cDNA, mRNA or other RNA, of synthetic origin, or any combination thereofcan encode such an isolated protein. Preferably, the isolated protein issubstantially free from proteins or polypeptides or other contaminantsthat are found in its natural environment that would interfere with itstherapeutic, diagnostic, prophylactic, research or other use.

The term “amino acid” includes its normal meaning in the art.

A “variant” of a polypeptide (e.g., an antigen binding protein, or anantibody) comprises an amino acid sequence wherein one or more aminoacid residues are inserted into, deleted from and/or substituted intothe amino acid sequence relative to another polypeptide sequence.Variants include fusion proteins.

The term “identity” refers to a relationship between the sequences oftwo or more polypeptide molecules or two or more nucleic acid molecules,as determined by aligning and comparing the sequences. “Percentidentity” means the percent of identical residues between the aminoacids or nucleotides in the compared molecules and is calculated basedon the size of the smallest of the molecules being compared. For thesecalculations, gaps in alignments (if any) are preferably addressed by aparticular mathematical model or computer program (i.e., an“algorithm”). Methods that can be used to calculate the identity of thealigned nucleic acids or polypeptides include those described inComputational Molecular Biology, (Lesk, A. M., ed.), 1988, New York:Oxford University Press; Biocomputing Informatics and Genome Projects,(Smith, D. W., ed.), 1993, New York: Academic Press; Computer Analysisof Sequence Data, Part I, (Griffin, A. M., and Griffin, H. G., eds.),1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysisin Molecular Biology, New York: Academic Press; Sequence AnalysisPrimer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M.Stockton Press; and Carillo et al., 1988, SIAM I Applied Math. 48:1073.

In calculating percent identity, the sequences being compared aretypically aligned in a way that gives the largest match between thesequences. One example of a computer program that can be used todetermine percent identity is the GCG program package, which includesGAP (Devereux et al., 1984, Nucl. Acid Res. 12:387; Genetics ComputerGroup, University of Wisconsin, Madison, Wis.). The computer algorithmGAP is used to align the two polypeptides or polynucleotides for whichthe percent sequence identity is to be determined. The sequences arealigned for optimal matching of their respective amino acid ornucleotide (the “matched span”, as determined by the algorithm). A gapopening penalty (which is calculated as 3× the average diagonal, whereinthe “average diagonal” is the average of the diagonal of the comparisonmatrix being used; the “diagonal” is the score or number assigned toeach perfect amino acid match by the particular comparison matrix) and agap extension penalty (which is usually 1/10 times the gap openingpenalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62are used in conjunction with the algorithm. In certain embodiments, astandard comparison matrix (see, Dayhoff et al., 1978, Atlas of ProteinSequence and Structure 5:345-352 for the PAM 250 comparison matrix;Henikoff et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919 forthe BLOSUM 62 comparison matrix) is also used by the algorithm.

Examples of parameters that can be employed in determining percentidentity for polypeptides or nucleotide sequences using the GAP programare the following:

-   -   Algorithm: Needleman et al., 1970, J. Mol. Biol. 48:443-453    -   Comparison matrix: BLOSUM 62 from Henikoff et al., 1992, supra    -   Gap Penalty: 12 (but with no penalty for end gaps)    -   Gap Length Penalty: 4    -   Threshold of Similarity: 0

Certain alignment schemes for aligning two amino acid sequences mayresult in matching of only a short region of the two sequences, and thissmall aligned region may have very high sequence identity even thoughthere is no significant relationship between the two full-lengthsequences. Accordingly, the selected alignment method (GAP program) canbe adjusted if so desired to result in an alignment that spans at least50 or other number of contiguous amino acids of the target polypeptide.

As used herein, the twenty conventional (e.g., naturally occurring)amino acids and their abbreviations follow conventional usage. SeeImmunology—A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds.,Sinauer Associates, Sunderland, Mass. (1991)), which is incorporatedherein by reference for any purpose. Stereoisomers (e.g., D-amino acids)of the twenty conventional amino acids, unnatural amino acids such asα-,α-disubstituted amino acids, N-alkyl amino acids, lactic acid, andother unconventional amino acids can also be suitable components forpolypeptides of the present invention. Examples of unconventional aminoacids include: 4-hydroxyproline, γ-carboxyglutamate,ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine,N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine,σ-N-methylarginine, and other similar amino acids and imino acids (e.g.,4-hydroxyproline). In the polypeptide notation used herein, theleft-hand direction is the amino terminal direction and the right-handdirection is the carboxy-terminal direction, in accordance with standardusage and convention.

Similarly, unless specified otherwise, the left-hand end ofsingle-stranded polynucleotide sequences is the 5′ end; the left-handdirection of double-stranded polynucleotide sequences is referred to asthe 5′ direction. The direction of 5′ to 3′ addition of nascent RNAtranscripts is referred to as the transcription direction; sequenceregions on the DNA strand having the same sequence as the RNA and whichare 5′ to the 5′ end of the RNA transcript are referred to as “upstreamsequences”; sequence regions on the DNA strand having the same sequenceas the RNA and which are 3′ to the 3′ end of the RNA transcript arereferred to as “downstream sequences.”

Conservative amino acid substitutions can encompass non-naturallyoccurring amino acid residues, which are typically incorporated bychemical peptide synthesis rather than by synthesis in biologicalsystems. These include peptidomimetics and other reversed or invertedforms of amino acid moieties.

Naturally occurring residues can be divided into classes based on commonside chain properties:

1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile;

2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

3) acidic: Asp, Glu;

4) basic: His, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Trp, Tyr, Phe.

For example, non-conservative substitutions can involve the exchange ofa member of one of these classes for a member from another class. Suchsubstituted residues can be introduced, for example, into regions of ahuman antibody that are homologous with non-human antibodies, or intothe non-homologous regions of the molecule.

In making changes to the antigen binding protein or the PCSK9 protein,according to certain embodiments, the hydropathic index of amino acidscan be considered. Each amino acid has been assigned a hydropathic indexon the basis of its hydrophobicity and charge characteristics. They are:isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9); and arginine(−4.5).

The importance of the hydropathic amino acid index in conferringinteractive biological function on a protein is understood in the art.Kyte et al., J. Mol. Biol., 157:105-131 (1982). It is known that certainamino acids can be substituted for other amino acids having a similarhydropathic index or score and still retain a similar biologicalactivity. In making changes based upon the hydropathic index, in certainembodiments, the substitution of amino acids whose hydropathic indicesare within ±2 is included. In certain embodiments, those which arewithin ±1 are included, and in certain embodiments, those within ±0.5are included.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity,particularly where the biologically functional protein or peptidethereby created is intended for use in immunological embodiments, as inthe present case. In certain embodiments, the greatest local averagehydrophilicity of a protein, as governed by the hydrophilicity of itsadjacent amino acids, correlates with its immunogenicity andantigenicity, i.e., with a biological property of the protein.

The following hydrophilicity values have been assigned to these aminoacid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1);glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);glycine (0); threonine (−0.4); proline (−0.5±1); alanine (−0.5);histidine (−0.5); cysteine (−1.0); methionine (−1.3); valine (−1.5);leucine (−1.8); isoleucine (−1.8); tyrosine (−2.3); phenylalanine (−2.5)and tryptophan (−3.4). In making changes based upon similarhydrophilicity values, in certain embodiments, the substitution of aminoacids whose hydrophilicity values are within ±2 is included, in certainembodiments, those which are within ±1 are included, and in certainembodiments, those within ±0.5 are included. One can also identifyepitopes from primary amino acid sequences on the basis ofhydrophilicity. These regions are also referred to as “epitopic coreregions.”

Exemplary amino acid substitutions are set forth in Table 1.

TABLE 1 Amino Acid Substitutions Original Exemplary Preferred ResiduesSubstitutions Substitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn LysAsn Gln Gln Asp Glu Glu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asp GlyPro, Ala Ala His Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Leu Phe,Norleucine Leu Norleucine, Ile, Ile Val, Met, Ala, Phe Lys Arg, 1,4Diamino-butyric Arg Acid, Gln, Asn Met Leu, Phe, Ile Leu Phe Leu, Val,Ile, Ala, Leu Tyr Pro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr,Phe Tyr Tyr Trp, Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Leu Ala,Norleucine

The term “derivative” refers to a molecule that includes a chemicalmodification other than an insertion, deletion, or substitution of aminoacids (or nucleic acids). In certain embodiments, derivatives comprisecovalent modifications, including, but not limited to, chemical bondingwith polymers, lipids, or other organic or inorganic moieties. Incertain embodiments, a chemically modified antigen binding protein canhave a greater circulating half-life than an antigen binding proteinthat is not chemically modified. In certain embodiments, a chemicallymodified antigen binding protein can have improved targeting capacityfor desired cells, tissues, and/or organs. In some embodiments, aderivative antigen binding protein is covalently modified to include oneor more water soluble polymer attachments, including, but not limitedto, polyethylene glycol, polyoxyethylene glycol, or polypropyleneglycol. See, e.g., U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144,4,670,417, 4,791,192 and 4,179,337. In certain embodiments, a derivativeantigen binding protein comprises one or more polymer, including, butnot limited to, monomethoxy-polyethylene glycol, dextran, cellulose, orother carbohydrate based polymers, poly-(N-vinylpyrrolidone)-polyethylene glycol, propylene glycol homopolymers, apolypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols(e.g., glycerol) and polyvinyl alcohol, as well as mixtures of suchpolymers.

In certain embodiments, a derivative is covalently modified withpolyethylene glycol (PEG) subunits. In certain embodiments, one or morewater-soluble polymer is bonded at one or more specific position, forexample at the amino terminus, of a derivative. In certain embodiments,one or more water-soluble polymer is randomly attached to one or moreside chains of a derivative. In certain embodiments, PEG is used toimprove the therapeutic capacity for an antigen binding protein. Incertain embodiments, PEG is used to improve the therapeutic capacity fora humanized antibody. Certain such methods are discussed, for example,in U.S. Pat. No. 6,133,426, which is hereby incorporated by referencefor any purpose.

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compound are termed “peptidemimetics” or “peptidomimetics.” Fauchere, J., Adv. Drug Res., 15:29(1986); Veber & Freidinger, TINS, p. 392 (1985); and Evans et al., J.Med. Chem., 30:1229 (1987), which are incorporated herein by referencefor any purpose. Such compounds are often developed with the aid ofcomputerized molecular modeling. Peptide mimetics that are structurallysimilar to therapeutically useful peptides can be used to produce asimilar therapeutic or prophylactic effect. Generally, peptidomimeticsare structurally similar to a paradigm polypeptide (i.e., a polypeptidethat has a biochemical property or pharmacological activity), such ashuman antibody, but have one or more peptide linkages optionallyreplaced by a linkage selected from: —CH₂NH—, —CH₂S—, —CH₂—CH₂—, —CH═CH—(cis and trans), —COCH₂—CH(OH)CH₂—, and —CH₂SO—, by methods well knownin the art. Systematic substitution of one or more amino acids of aconsensus sequence with a D-amino acid of the same type (e.g., D-lysinein place of L-lysine) can be used in certain embodiments to generatemore stable peptides. In addition, constrained peptides comprising aconsensus sequence or a substantially identical consensus sequencevariation can be generated by methods known in the art (Rizo andGierasch, Ann. Rev. Biochem., 61:387 (1992), incorporated herein byreference for any purpose); for example, by adding internal cysteineresidues capable of forming intramolecular disulfide bridges whichcyclize the peptide.

The term “naturally occurring” as used throughout the specification inconnection with biological materials such as polypeptides, nucleicacids, host cells, and the like, refers to materials which are found innature or a form of the materials that is found in nature.

An “antigen binding protein” (“ABP”) as used herein means any proteinthat binds a specified target antigen. In the instant application, thespecified target antigen is the PCSK9 protein or fragment thereof“Antigen binding protein” includes but is not limited to antibodies andbinding parts thereof, such as immunologically functional fragments.Peptibodies are another example of antigen binding proteins. The term“immunologically functional fragment” (or simply “fragment”) of anantibody or immunoglobulin chain (heavy or light chain) antigen bindingprotein, as used herein, is a species of antigen binding proteincomprising a portion (regardless of how that portion is obtained orsynthesized) of an antibody that lacks at least some of the amino acidspresent in a full-length chain but which is still capable ofspecifically binding to an antigen. Such fragments are biologicallyactive in that they bind to the target antigen and can compete withother antigen binding proteins, including intact antibodies, for bindingto a given epitope. In some embodiments, the fragments are neutralizingfragments. In some embodiments, the fragments can block or reduce thelikelihood of the interaction between LDLR and PCSK9. In one aspect,such a fragment will retain at least one CDR present in the full-lengthlight or heavy chain, and in some embodiments will comprise a singleheavy chain and/or light chain or portion thereof. These biologicallyactive fragments can be produced by recombinant DNA techniques, or canbe produced by enzymatic or chemical cleavage of antigen bindingproteins, including intact antibodies. Immunologically functionalimmunoglobulin fragments include, but are not limited to, Fab, a diabody(heavy chain variable domain on the same polypeptide as a light chainvariable domain, connected via a short peptide linker that is too shortto permit pairing between the two domains on the same chain), Fab′,F(ab′)₂, Fv, domain antibodies and single-chain antibodies, and can bederived from any mammalian source, including but not limited to human,mouse, rat, camelid or rabbit. It is further contemplated that afunctional portion of the antigen binding proteins disclosed herein, forexample, one or more CDRs, could be covalently bound to a second proteinor to a small molecule to create a therapeutic agent directed to aparticular target in the body, possessing bifunctional therapeuticproperties, or having a prolonged serum half-life. As will beappreciated by one of skill in the art, an antigen binding protein caninclude nonprotein components. In some sections of the presentdisclosure, examples of ABPs are described herein in terms of“number/letter/number” (e.g., 25A7). In these cases, the exact namedenotes a specific antibody. That is, an ABP named 25A7 is notnecessarily the same as an antibody named 25A7.1, (unless they areexplicitly taught as the same in the specification, e.g., 25A7 and25A7.3). As will be appreciated by one of skill in the art, in someembodiments LDLR is not an antigen binding protein. In some embodiments,binding subsections of LDLR are not antigen binding proteins, e.g.,EGFa. In some embodiments, other molecules through which PCSK9 signalsin vivo are not antigen binding proteins. Such embodiments will beexplicitly identified as such.

Certain antigen binding proteins described herein are antibodies or arederived from antibodies. In certain embodiments, the polypeptidestructure of the antigen binding proteins is based on antibodies,including, but not limited to, monoclonal antibodies, bispecificantibodies, minibodies, domain antibodies, synthetic antibodies(sometimes referred to herein as “antibody mimetics”), chimericantibodies, humanized antibodies, human antibodies, antibody fusions(sometimes referred to herein as “antibody conjugates”), and fragmentsthereof, respectively. In some embodiments, the ABP comprises orconsists of avimers (tightly binding peptide). These various antigenbinding proteins are further described herein.

An “Fc” region comprises two heavy chain fragments comprising the C_(H)1and C_(H)2 domains of an antibody. The two heavy chain fragments areheld together by two or more disulfide bonds and by hydrophobicinteractions of the C_(H)3 domains.

A “Fab fragment” comprises one light chain and the C_(H)1 and variableregions of one heavy chain. The heavy chain of a Fab molecule cannotform a disulfide bond with another heavy chain molecule.

A “Fab′ fragment” comprises one light chain and a portion of one heavychain that contains the VH domain and the C_(H)1 domain and also theregion between the C_(H)1 and C_(H)2 domains, such that an interchaindisulfide bond can be formed between the two heavy chains of two Fab′fragments to form an F(ab′)₂ molecule.

A “F(ab′)₂ fragment” contains two light chains and two heavy chainscontaining a portion of the constant region between the C_(H)1 andC_(H)2 domains, such that an interchain disulfide bond is formed betweenthe two heavy chains. A F(ab′)₂ fragment thus is composed of two Fab′fragments that are held together by a disulfide bond between the twoheavy chains.

The “Fv region” comprises the variable regions from both the heavy andlight chains, but lacks the constant regions.

“Single-chain antibodies” are Fv molecules in which the heavy and lightchain variable regions have been connected by a flexible linker to forma single polypeptide chain, which forms an antigen binding region.Single chain antibodies are discussed in detail in International PatentApplication Publication No. WO 88/01649 and U.S. Pat. No. 4,946,778 andU.S. Pat. No. 5,260,203, the disclosures of which are incorporated byreference.

A “domain antibody” is an immunologically functional immunoglobulinfragment containing only the variable region of a heavy chain or thevariable region of a light chain. In some instances, two or more V_(H)regions are covalently joined with a peptide linker to create a bivalentdomain antibody. The two V_(H) regions of a bivalent domain antibody cantarget the same or different antigens.

A “bivalent antigen binding protein” or “bivalent antibody” comprisestwo antigen binding sites. In some instances, the two binding sites havethe same antigen specificities. Bivalent antigen binding proteins andbivalent antibodies can be bispecific, see, infra. A bivalent antibodyother than a “multispecific” or “multifunctional” antibody, in certainembodiments, typically is understood to have each of its binding sitesidentical.

A “multispecific antigen binding protein” or “multispecific antibody” isone that targets more than one antigen or epitope.

A “bispecific,” “dual-specific” or “bifunctional” antigen bindingprotein or antibody is a hybrid antigen binding protein or antibody,respectively, having two different antigen binding sites. Bispecificantigen binding proteins and antibodies are a species of multispecificantigen binding protein antibody and can be produced by a variety ofmethods including, but not limited to, fusion of hybridomas or linkingof Fab′ fragments. See, e.g., Songsivilai and Lachmann, 1990, Clin. Exp.Immunol. 79:315-321; Kostelny et al., 1992, J. Immunol. 148:1547-1553.The two binding sites of a bispecific antigen binding protein orantibody will bind to two different epitopes, which can reside on thesame or different protein targets.

An antigen binding protein is said to “specifically bind” its targetantigen when the dissociation constant (K_(d)) is ≦10⁻⁷ M. The ABPspecifically binds antigen with “high affinity” when the K_(d) is≦5×10⁻⁹ M, and with “very high affinity” when the K_(d) is ≦5×10⁻¹⁰ M.In one embodiment, the ABP has a K_(d) of ≦10⁻⁹ M. In one embodiment,the off-rate is <1×10⁻⁵. In other embodiments, the ABPs will bind tohuman PCSK9 with a K_(d) of between about 10⁻⁹ M and 10⁻¹³ M, and in yetanother embodiment the ABPs will bind with a K_(d)≦5×10⁻¹⁰. As will beappreciated by one of skill in the art, in some embodiments, any or allof the antigen binding fragments can specifically bind to PCSK9.

An antigen binding protein is “selective” when it binds to one targetmore tightly than it binds to a second target.

“Antigen binding region” means a protein, or a portion of a protein,that specifically binds a specified antigen (e.g., a paratope). Forexample, that portion of an antigen binding protein that contains theamino acid residues that interact with an antigen and confer on theantigen binding protein its specificity and affinity for the antigen isreferred to as “antigen binding region.” An antigen binding regiontypically includes one or more “complementary binding regions” (“CDRs”).Certain antigen binding regions also include one or more “framework”regions. A “CDR” is an amino acid sequence that contributes to antigenbinding specificity and affinity. “Framework” regions can aid inmaintaining the proper conformation of the CDRs to promote bindingbetween the antigen binding region and an antigen. Structurally,framework regions can be located in antibodies between CDRs. Examples offramework and CDR regions are shown in FIGS. 2A-3D, 3CCC-3JJJ, and15A-15D. In some embodiments, the sequences for CDRs for the light chainof antibody 3B6 are as follows: CDR1 TLSSGYSSYEVD (SEQ ID NO: 279);CDR2VDTGGIVGSKGE (SEQ ID NO: 280); CDR3 GADHGSGTNFVVV (SEQ ID NO: 281),and the FRs are as follows: FR1 QPVLTQPLFASASLGASVTLTC (SEQ ID NO: 282);FR2WYQQRPGKGPRFVMR (SEQ ID NO: 283); FR3GIPDRFSVLGSGLNRYLTIKNIQEEDESDYHC(SEQ ID NO: 284); and FR4FGGGTKLTVL (SEQ ID NO: 285).

In certain aspects, recombinant antigen binding proteins that bindPCSK9, for example human PCSK9, are provided. In this context, a“recombinant antigen binding protein” is a protein made usingrecombinant techniques, i.e., through the expression of a recombinantnucleic acid as described herein. Methods and techniques for theproduction of recombinant proteins are well known in the art.

The term “antibody” refers to an intact immunoglobulin of any isotype,or a fragment thereof that can compete with the intact antibody forspecific binding to the target antigen, and includes, for instance,chimeric, humanized, fully human, and bispecific antibodies. An“antibody” is a species of an antigen binding protein. An intactantibody will generally comprise at least two full-length heavy chainsand two full-length light chains, but in some instances can includefewer chains such as antibodies naturally occurring in camelids whichcan comprise only heavy chains. Antibodies can be derived solely from asingle source, or can be “chimeric,” that is, different portions of theantibody can be derived from two different antibodies as describedfurther below. The antigen binding proteins, antibodies, or bindingfragments can be produced in hybridomas, by recombinant DNA techniques,or by enzymatic or chemical cleavage of intact antibodies. Unlessotherwise indicated, the term “antibody” includes, in addition toantibodies comprising two full-length heavy chains and two full-lengthlight chains, derivatives, variants, fragments, and muteins thereof,examples of which are described below. Furthermore, unless explicitlyexcluded, antibodies include monoclonal antibodies, bispecificantibodies, minibodies, domain antibodies, synthetic antibodies(sometimes referred to herein as “antibody mimetics”), chimericantibodies, humanized antibodies, human antibodies, antibody fusions(sometimes referred to herein as “antibody conjugates”), and fragmentsthereof, respectively. In some embodiments, the term also encompassespeptibodies.

Naturally occurring antibody structural units typically comprise atetramer. Each such tetramer typically is composed of two identicalpairs of polypeptide chains, each pair having one full-length “light”(in certain embodiments, about 25 kDa) and one full-length “heavy” chain(in certain embodiments, about 50-70 kDa). The amino-terminal portion ofeach chain typically includes a variable region of about 100 to 110 ormore amino acids that typically is responsible for antigen recognition.The carboxy-terminal portion of each chain typically defines a constantregion that can be responsible for effector function. Human light chainsare typically classified as kappa and lambda light chains. Heavy chainsare typically classified as mu, delta, gamma, alpha, or epsilon, anddefine the antibody's isotype as IgM, IgD, IgG, IgA, and IgE,respectively. IgG has several subclasses, including, but not limited to,IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including, but notlimited to, IgM1 and IgM2. IgA is similarly subdivided into subclassesincluding, but not limited to, IgA1 and IgA2. Within full-length lightand heavy chains, typically, the variable and constant regions arejoined by a “J” region of about 12 or more amino acids, with the heavychain also including a “D” region of about 10 more amino acids. See,e.g., Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd ed. Raven Press,N.Y. (1989)) (incorporated by reference in its entirety for allpurposes). The variable regions of each light/heavy chain pair typicallyform the antigen binding site.

The variable regions typically exhibit the same general structure ofrelatively conserved framework regions (FR) joined by three hypervariable regions, also called complementarity determining regions orCDRs. The CDRs from the two chains of each pair typically are aligned bythe framework regions, which can enable binding to a specific epitope.From N-terminal to C-terminal, both light and heavy chain variableregions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3and FR4. The assignment of amino acids to each domain is typically inaccordance with the definitions of Kabat Sequences of Proteins ofImmunological Interest (National Institutes of Health, Bethesda, Md.(1987 and 1991)), or Chothia & Lesk, J. Mol. Biol., 196:901-917 (1987);Chothia et al., Nature, 342:878-883 (1989).

In certain embodiments, an antibody heavy chain binds to an antigen inthe absence of an antibody light chain. In certain embodiments, anantibody light chain binds to an antigen in the absence of an antibodyheavy chain. In certain embodiments, an antibody binding region binds toan antigen in the absence of an antibody light chain. In certainembodiments, an antibody binding region binds to an antigen in theabsence of an antibody heavy chain. In certain embodiments, anindividual variable region specifically binds to an antigen in theabsence of other variable regions.

In certain embodiments, definitive delineation of a CDR andidentification of residues comprising the binding site of an antibody isaccomplished by solving the structure of the antibody and/or solving thestructure of the antibody-ligand complex. In certain embodiments, thatcan be accomplished by any of a variety of techniques known to thoseskilled in the art, such as X-ray crystallography. In certainembodiments, various methods of analysis can be employed to identify orapproximate the CDR regions. Examples of such methods include, but arenot limited to, the Kabat definition, the Chothia definition, the AbMdefinition and the contact definition.

The Kabat definition is a standard for numbering the residues in anantibody and is typically used to identify CDR regions. See, e.g.,Johnson & Wu, Nucleic Acids Res., 28: 214-8 (2000). The Chothiadefinition is similar to the Kabat definition, but the Chothiadefinition takes into account positions of certain structural loopregions. See, e.g., Chothia et al., J. Mol. Biol., 196: 901-17 (1986);Chothia et al., Nature, 342: 877-83 (1989). The AbM definition uses anintegrated suite of computer programs produced by Oxford Molecular Groupthat model antibody structure. See, e.g., Martin et al., Proc Natl AcadSci (USA), 86:9268-9272 (1989); “AbM™, A Computer Program for ModelingVariable Regions of Antibodies,” Oxford, UK; Oxford Molecular, Ltd. TheAbM definition models the tertiary structure of an antibody from primarysequence using a combination of knowledge databases and ab initiomethods, such as those described by Samudrala et al., “Ab Initio ProteinStructure Prediction Using a Combined Hierarchical Approach,” inPROTEINS, Structure, Function and Genetics Suppl., 3:194-198 (1999). Thecontact definition is based on an analysis of the available complexcrystal structures. See, e.g., MacCallum et al., J. Mol. Biol., 5:732-45(1996).

By convention, the CDR regions in the heavy chain are typically referredto as H1, H2, and H3 and are numbered sequentially in the direction fromthe amino terminus to the carboxy terminus. The CDR regions in the lightchain are typically referred to as L1, L2, and L3 and are numberedsequentially in the direction from the amino terminus to the carboxyterminus.

The term “light chain” includes a full-length light chain and fragmentsthereof having sufficient variable region sequence to confer bindingspecificity. A full-length light chain includes a variable regiondomain, V_(L), and a constant region domain, C_(L). The variable regiondomain of the light chain is at the amino-terminus of the polypeptide.Light chains include kappa chains and lambda chains.

The term “heavy chain” includes a full-length heavy chain and fragmentsthereof having sufficient variable region sequence to confer bindingspecificity. A full-length heavy chain includes a variable regiondomain, V_(H), and three constant region domains, C_(H)1, C_(H)2, andC_(H)3. The V_(H) domain is at the amino-terminus of the polypeptide,and the C_(H) domains are at the carboxyl-terminus, with the C_(H)3being closest to the carboxy-terminus of the polypeptide. Heavy chainscan be of any isotype, including IgG (including IgG1, IgG2, IgG3 andIgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM and IgE.

A bispecific or bifunctional antibody typically is an artificial hybridantibody having two different heavy/light chain pairs and two differentbinding sites. Bispecific antibodies can be produced by a variety ofmethods including, but not limited to, fusion of hybridomas or linkingof Fab′ fragments. See, e.g., Songsivilai et al., Clin. Exp. Immunol.,79: 315-321 (1990); Kostelny et al., J. Immunol., 148:1547-1553 (1992).

Some species of mammals also produce antibodies having only a singleheavy chain.

Each individual immunoglobulin chain is typically composed of several“immunoglobulin domains,” each consisting of roughly 90 to 110 aminoacids and having a characteristic folding pattern. These domains are thebasic units of which antibody polypeptides are composed. In humans, theIgA and IgD isotypes contain four heavy chains and four light chains;the IgG and IgE isotypes contain two heavy chains and two light chains;and the IgM isotype contains five heavy chains and five light chains.The heavy chain C region typically comprises one or more domains thatcan be responsible for effector function. The number of heavy chainconstant region domains will depend on the isotype. IgG heavy chains,for example, contain three C region domains known as C_(H)1, C_(H)2 andC_(H)3. The antibodies that are provided can have any of these isotypesand subtypes. In certain embodiments of the present invention, ananti-PCSK9 antibody is of the IgG2 or IgG4 subtype.

The term “variable region” or “variable domain” refers to a portion ofthe light and/or heavy chains of an antibody, typically includingapproximately the amino-terminal 120 to 130 amino acids in the heavychain and about 100 to 110 amino terminal amino acids in the lightchain. In certain embodiments, variable regions of different antibodiesdiffer extensively in amino acid sequence even among antibodies of thesame species. The variable region of an antibody typically determinesspecificity of a particular antibody for its target

The term “neutralizing antigen binding protein” or “neutralizingantibody” refers to an antigen binding protein or antibody,respectively, that binds to a ligand and prevents or reduces thebiological effect of that ligand. This can be done, for example, bydirectly blocking a binding site on the ligand or by binding to theligand and altering the ligand's ability to bind through indirect means(such as structural or energetic alterations in the ligand). In someembodiments, the term can also denote an antigen binding protein thatprevents the protein to which it is bound from performing a biologicalfunction. In assessing the binding and/or specificity of an antigenbinding protein, e.g., an antibody or immunologically functionalfragment thereof, an antibody or fragment can substantially inhibitbinding of a ligand to its binding partner when an excess of antibodyreduces the quantity of binding partner bound to the ligand by at leastabout 1-20, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-85%,85-90%, 90-95%, 95-97%, 97-98%, 98-99% or more (as measured in an invitro competitive binding assay). In some embodiments, in the case ofPCSK9 antigen binding proteins, such a neutralizing molecule candiminish the ability of PCSK9 to bind the LDLR. In some embodiments, theneutralizing ability is characterized and/or described via a competitionassay. In some embodiments, the neutralizing ability is described interms of an IC₅₀ or EC₅₀ value. In some embodiments, ABPs 27B2, 13H1,13B5 and 3C4 are non-neutralizing ABPs, 3B6, 9C9 and 31A4 are weakneutralizers, and the remaining ABPs in Table 2 are strong neutralizers.In some embodiments, the antibodies or antigen binding proteinsneutralize by binding to PCSK9 and preventing PCSK9 from binding to LDLR(or reducing the ability of PCSK9 to bind to LDLR). In some embodiments,the antibodies or ABPs neutralize by binding to PCSK9, and while stillallowing PCSK9 to bind to LDLR, preventing or reducing the PCSK9mediated degradation of LDLR. Thus, in some embodiments, a neutralizingABP or antibody can still permit PCSK9/LDLR binding, but will prevent(or reduce) subsequent PCSK9 involved degradation of LDLR.

The term “target” refers to a molecule or a portion of a moleculecapable of being bound by an antigen binding protein. In certainembodiments, a target can have one or more epitopes. In certainembodiments, a target is an antigen. The use of “antigen” in the phrase“antigen binding protein” simply denotes that the protein sequence thatcomprises the antigen can be bound by an antibody. In this context, itdoes not require that the protein be foreign or that it be capable ofinducing an immune response.

The term “compete” when used in the context of antigen binding proteins(e.g., neutralizing antigen binding proteins or neutralizing antibodies)that compete for the same epitope means competition between antigenbinding proteins as determined by an assay in which the antigen bindingprotein (e.g., antibody or immunologically functional fragment thereof)being tested prevents or inhibits (e.g., reduces) specific binding of areference antigen binding protein (e.g., a ligand, or a referenceantibody) to a common antigen (e.g., PCSK9 or a fragment thereof).Numerous types of competitive binding assays can be used to determine ifone antigen binding protein competes with another, for example: solidphase direct or indirect radioimmunoassay (MA), solid phase direct orindirect enzyme immunoassay (EIA), sandwich competition assay (see,e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253); solid phasedirect biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol.137:3614-3619) solid phase direct labeled assay, solid phase directlabeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, ALaboratory Manual, Cold Spring Harbor Press); solid phase direct labelRIA using 1-125 label (see, e.g., Morel et al., 1988, Molec. Immunol.25:7-15); solid phase direct biotin-avidin EIA (see, e.g., Cheung, etal., 1990, Virology 176:546-552); and direct labeled RIA (Moldenhauer etal., 1990, Scand. J. Immunol. 32:77-82). Typically, such an assayinvolves the use of purified antigen bound to a solid surface or cellsbearing either of these, an unlabelled test antigen binding protein anda labeled reference antigen binding protein. Competitive inhibition ismeasured by determining the amount of label bound to the solid surfaceor cells in the presence of the test antigen binding protein. Usuallythe test antigen binding protein is present in excess. Antigen bindingproteins identified by competition assay (competing antigen bindingproteins) include antigen binding proteins binding to the same epitopeas the reference antigen binding proteins and antigen binding proteinsbinding to an adjacent epitope sufficiently proximal to the epitopebound by the reference antigen binding protein for steric hindrance tooccur. Additional details regarding methods for determining competitivebinding are provided in the examples herein. Usually, when a competingantigen binding protein is present in excess, it will inhibit (e.g.,reduce) specific binding of a reference antigen binding protein to acommon antigen by at least 40-45%, 45-50%, 50-55%, 55-60%, 60-65%,65-70%, 70-75% or 75% or more. In some instances, binding is inhibitedby at least 80-85%, 85-90%, 90-95%, 95-97%, or 97% or more.

The term “antigen” refers to a molecule or a portion of a moleculecapable of being bound by a selective binding agent, such as an antigenbinding protein (including, e.g., an antibody or immunologicalfunctional fragment thereof). In some embodiments, the antigen iscapable of being used in an animal to produce antibodies capable ofbinding to that antigen. An antigen can possess one or more epitopesthat are capable of interacting with different antigen binding proteins,e.g., antibodies.

The term “epitope” includes any determinant capable being bound by anantigen binding protein, such as an antibody or to a T-cell receptor. Anepitope is a region of an antigen that is bound by an antigen bindingprotein that targets that antigen, and when the antigen is a protein,includes specific amino acids that directly contact the antigen bindingprotein. Most often, epitopes reside on proteins, but in some instancescan reside on other kinds of molecules, such as nucleic acids. Epitopedeterminants can include chemically active surface groupings ofmolecules such as amino acids, sugar side chains, phosphoryl or sulfonylgroups, and can have specific three dimensional structuralcharacteristics, and/or specific charge characteristics. Generally,antibodies specific for a particular target antigen will preferentiallyrecognize an epitope on the target antigen in a complex mixture ofproteins and/or macromolecules.

As used herein, “substantially pure” means that the described species ofmolecule is the predominant species present, that is, on a molar basisit is more abundant than any other individual species in the samemixture. In certain embodiments, a substantially pure molecule is acomposition wherein the object species comprises at least 50% (on amolar basis) of all macromolecular species present. In otherembodiments, a substantially pure composition will comprise at least80%, 85%, 90%, 95%, or 99% of all macromolecular species present in thecomposition. In other embodiments, the object species is purified toessential homogeneity wherein contaminating species cannot be detectedin the composition by conventional detection methods and thus thecomposition consists of a single detectable macromolecular species.

The term “agent” is used herein to denote a chemical compound, a mixtureof chemical compounds, a biological macromolecule, or an extract madefrom biological materials.

As used herein, the terms “label” or “labeled” refers to incorporationof a detectable marker, e.g., by incorporation of a radiolabeled aminoacid or attachment to a polypeptide of biotin moieties that can bedetected by marked avidin (e.g., streptavidin containing a fluorescentmarker or enzymatic activity that can be detected by optical orcolorimetric methods). In certain embodiments, the label or marker canalso be therapeutic. Various methods of labeling polypeptides andglycoproteins are known in the art and can be used. Examples of labelsfor polypeptides include, but are not limited to, the following:radioisotopes or radionuclides (e.g., ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc,¹¹¹In, ¹²⁵I, ¹³¹I) fluorescent labels (e.g., FITC, rhodamine, lanthanidephosphors), enzymatic labels (e.g., horseradish peroxidase,β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent,biotinyl groups, predetermined polypeptide epitopes recognized by asecondary reporter (e.g., leucine zipper pair sequences, binding sitesfor secondary antibodies, metal binding domains, epitope tags). Incertain embodiments, labels are attached by spacer arms of variouslengths to reduce potential steric hindrance.

The term “biological sample”, as used herein, includes, but is notlimited to, any quantity of a substance from a living thing or formerlyliving thing. Such living things include, but are not limited to,humans, mice, monkeys, rats, rabbits, and other animals. Such substancesinclude, but are not limited to, blood, serum, urine, cells, organs,tissues, bone, bone marrow, lymph nodes, and skin.

The term “pharmaceutical agent composition” (or agent or drug) as usedherein refers to a chemical compound, composition, agent or drug capableof inducing a desired therapeutic effect when properly administered to apatient. It does not necessarily require more than one type ofingredient.

The term “therapeutically effective amount” refers to the amount of aPCSK9 antigen binding protein determined to produce a therapeuticresponse in a mammal. Such therapeutically effective amounts are readilyascertained by one of ordinary skill in the art.

The term “modulator,” as used herein, is a compound that changes oralters the activity or function of a molecule. For example, a modulatorcan cause an increase or decrease in the magnitude of a certain activityor function of a molecule compared to the magnitude of the activity orfunction observed in the absence of the modulator. In certainembodiments, a modulator is an inhibitor, which decreases the magnitudeof at least one activity or function of a molecule. Certain exemplaryactivities and functions of a molecule include, but are not limited to,binding affinity, enzymatic activity, and signal transduction. Certainexemplary inhibitors include, but are not limited to, proteins,peptides, antibodies, peptibodies, carbohydrates or small organicmolecules. Peptibodies are described in, e.g., U.S. Pat. No. 6,660,843(corresponding to PCT Application No. WO 01/83525).

The terms “patient” and “subject” are used interchangeably and includehuman and non-human animal subjects as well as those with formallydiagnosed disorders, those without formally recognized disorders, thosereceiving medical attention, those at risk of developing the disorders,etc.

The term “treat” and “treatment” includes therapeutic treatments,prophylactic treatments, and applications in which one reduces the riskthat a subject will develop a disorder or other risk factor. Treatmentdoes not require the complete curing of a disorder and encompassesembodiments in which one reduces symptoms or underlying risk factors.

The term “prevent” does not require the 100% elimination of thepossibility of an event. Rather, it denotes that the likelihood of theoccurrence of the event has been reduced in the presence of the compoundor method.

Standard techniques can be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques can beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures can be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See, e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989)), which is incorporated herein by referencefor any purpose. Unless specific definitions are provided, thenomenclatures utilized in connection with, and the laboratory proceduresand techniques of, analytical chemistry, synthetic organic chemistry,and medicinal and pharmaceutical chemistry described herein are thosewell known and commonly used in the art. Standard techniques can be usedfor chemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

Antigen Binding Proteins to PCSK9

Proprotein convertase subtilisin kexin type 9 (PCSK9) is a serineprotease involved in regulating the levels of the low densitylipoprotein receptor (LDLR) protein (Horton et al., 2007; Seidah andPrat, 2007). PCSK9 is a prohormone-proprotein convertase in thesubtilisin (S8) family of serine proteases (Seidah et al., 2003). Anexemplary human PCSK9 amino acid sequence is presented as SEQ ID NOs: 1and 3. in FIG. 1A (depicting the “pro” domain of the protein asunderlined) and FIG. 1B (depicting the signal sequence in bold and thepro domain underlined). An exemplary human PCSK9 coding sequence ispresented as SEQ ID NO: 2 (FIG. 1B). As described herein, PCSK9 proteinscan also include fragments of the full length PCSK9 protein. Thestructure of the PCSK9 protein has recently been solved by two groups(Cunningham et al., Nature Structural & Molecular Biology, 2007, andPiper et al., Structure, 15:1-8, 2007), the entireties of both of whichare herein incorporated by reference. PCSK9 includes a signal sequence,a N-terminal prodomain, a subtilisin-like catalytic domain and aC-terminal domain.

Antigen binding proteins (ABPs) that bind PCSK9, including human PCSK9,are provided herein. In some embodiments, the antigen binding proteinsprovided are polypeptides which comprise one or more complementarydetermining regions (CDRs), as described herein. In some antigen bindingproteins, the CDRs are embedded into a “framework” region, which orientsthe CDR(s) such that the proper antigen binding properties of the CDR(s)is achieved. In some embodiments, antigen binding proteins providedherein can interfere with, block, reduce or modulate the interactionbetween PCSK9 and LDLR. Such antigen binding proteins are denoted as“neutralizing.” In some embodiments, binding between PCSK9 and LDLR canstill occur, even though the antigen binding protein is neutralizing andbound to PCSK9. For example, in some embodiments, the ABP prevents orreduces the adverse influence of PCSK9 on LDLR without blocking the LDLRbinding site on PCSK9. Thus, in some embodiments, the ABP modulates oralters PCSK9's ability to result in the degradation of LDLR, withouthaving to prevent the binding interaction between PCSK9 and LDLR. SuchABPs can be specifically described as “non-competitively neutralizing”ABPs. In some embodiments, the neutralizing ABP binds to PCSK9 in alocation and/or manner that prevents PCSK9 from binding to LDLR. SuchABPs can be specifically described as “competitively neutralizing” ABPs.Both of the above neutralizers can result in a greater amount of freeLDLR being present in a subject, which results in more LDLR binding toLDL (thereby reducing the amount of LDL in the subject). In turn, thisresults in a reduction in the amount of serum cholesterol present in asubject.

In some embodiments, the antigen binding proteins provided herein arecapable of inhibiting PCSK9-mediated activity (including binding). Insome embodiments, antigen binding proteins binding to these epitopesinhibit, inter alia, interactions between PCSK9 and LDLR and otherphysiological effects mediated by PCSK9. In some embodiments, theantigen binding proteins are human, such as fully human antibodies toPCSK9.

In some embodiments, the ABP binds to the catalytic domain of PCSK9. Insome embodiments, the ABP binds to the mature form of PCSK9. In someembodiments the ABP binds in the prodomain of PCSK9. In someembodiments, the ABP selectively binds to the mature form of PCSK9. Insome embodiments, the ABP binds to the catalytic domain in a manner suchthat PCSK9 cannot bind or bind as efficiently to LDLR. In someembodiments, the antigen binding protein does not bind to the c-terminusof the cataylytic domain. In some embodiments, the antigen bindingprotein does not bind to the n-terminus of the catalytic domain. In someembodiments, the ABP does not bind to the n- or c-terminus of the PCSK9protein. In some embodiments, the ABP binds to any one of the epitopesbound by the antibodies discussed herein. In some embodiments, this canbe determined by competition assays between the antibodies disclosedherein and other antibodies. In some embodiments, the ABP binds to anepitope bound by one of the antibodies described in Table 2. In someembodiments, the antigen binding proteins bind to a specificconformational state of PCSK9 so as to prevent PCSK9 from interactingwith LDLR. In some embodiments, the ABP binds to the V domain of PCSK9.In some embodiments, the ABP binds to the V domain of PCSK9 and prevents(or reduces) PCSK9 from binding to LDLR. In some embodiments, the ABPbinds to the V domain of PCSK9, and while it does not prevent (orreduce) the binding of PCSK9 to LDLR, the ABP prevents or reduces theadverse activities mediated through PCSK9 on LDLR.

The antigen binding proteins that are disclosed herein have a variety ofutilities. Some of the antigen binding proteins, for instance, areuseful in specific binding assays, affinity purification of PCSK9, inparticular human PCSK9 or its ligands and in screening assays toidentify other antagonists of PCSK9 activity. Some of the antigenbinding proteins are useful for inhibiting binding of PCSK9 to LDLR, orinhibiting PCSK9-mediated activities.

The antigen binding proteins can be used in a variety of therapeuticapplications, as explained herein. For example, in some embodiments thePCSK9 antigen binding proteins are useful for treating conditionsassociated with PCSK9, such as cholesterol related disorders (or “serumcholesterol related disorders”) such as hypercholesterolemia, as furtherdescribed herein. Other uses for the antigen binding proteins include,for example, diagnosis of PCSK9-associated diseases or conditions andscreening assays to determine the presence or absence of PCSK9. Some ofthe antigen binding proteins described herein are useful in treatingconsequences, symptoms, and/or the pathology associated with PCSK9activity.

In some embodiments, the antigen binding proteins that are providedcomprise one or more CDRs (e.g., 1, 2, 3, 4, 5 or 6 CDRs). In someembodiments, the antigen binding protein comprises (a) a polypeptidestructure and (b) one or more CDRs that are inserted into and/or joinedto the polypeptide structure. The polypeptide structure can take avariety of different forms. For example, it can be, or comprise, theframework of a naturally occurring antibody, or fragment or variantthereof, or can be completely synthetic in nature. Examples of variouspolypeptide structures are further described below.

In certain embodiments, the polypeptide structure of the antigen bindingproteins is an antibody or is derived from an antibody, including, butnot limited to, monoclonal antibodies, bispecific antibodies,minibodies, domain antibodies, synthetic antibodies (sometimes referredto herein as “antibody mimetics”), chimeric antibodies, humanizedantibodies, antibody fusions (sometimes referred to as “antibodyconjugates”), and portions or fragments of each, respectively. In someinstances, the antigen binding protein is an immunological fragment ofan antibody (e.g., a Fab, a Fab′, a F(ab′)₂, or a scFv). The variousstructures are further described and defined herein.

Certain of the antigen binding proteins as provided herein specificallyand/or selectively bind to human PCSK9. In some embodiments, the antigenbinding protein specifically and/or selectively binds to human PCSK9protein having and/or consisting of residues 153-692 of SEQ ID NO: 3. Insome embodiments the ABP specifically and/or selectively binds to humanPCSK9 having and/or consisting of residues 31-152 of SEQ ID NO: 3. Insome embodiments, the ABP selectively binds to a human PCSK9 protein asdepicted in FIG. 1A (SEQ ID NO: 1). In some embodiments, the antigenbinding protein specifically binds to at least a fragment of the PCSK9protein and/or a full length PCSK9 protein, with or without a signalsequence.

In embodiments where the antigen binding protein is used for therapeuticapplications, an antigen binding protein can inhibit, interfere with ormodulate one or more biological activities of PCSK9. In one embodiment,an antigen binding protein binds specifically to human PCSK9 and/orsubstantially inhibits binding of human PCSK9 to LDLR by at least about20%-40%, 40-60%, 60-80%, 80-85%, or more (for example, by measuringbinding in an in vitro competitive binding assay). Some of the antigenbinding proteins that are provided herein are antibodies. In someembodiments, the ABP has a K_(d) of less (binding more tightly) than10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹³ M. In some embodiments, theABP has an IC₅₀ for blocking the binding of LDLR to PCSK9 (D374Y, highaffinity variant) of less than 1 microM, 1000 nM to 100 nM, 100 nM to 10nM, 10 nM to 1 nM, 1000 pM to 500 pM, 500 pM to 200 pM, less than 200pM, 200 pM to 150 pM, 200 pM to 100 pM, 100 pM to 10 pM, 10 pM to 1 pM.

One example of an IgG2 heavy chain constant domain of an anti-PCSK9antibody of the present invention has the amino acid sequence as shownin SEQ ID NO: 154, FIG. 3KK.

One example of an IgG4 heavy chain constant domain of an anti-PCSK9antibody of the present invention has the amino acid sequence as shownin SEQ ID NO: 155, FIG. 3KK.

One example of a kappa light chain constant domain of an anti-PCSK9antibody has the amino acid sequence as shown in SEQ ID NO: 157, FIG.3KK.

One example of a lambda light chain constant domain of an anti-PCSK9antibody has the amino acid sequence as shown in SEQ ID NO: 156, FIG.3KK.

Variable regions of immunoglobulin chains generally exhibit the sameoverall structure, comprising relatively conserved framework regions(FR) joined by three hypervariable regions, more often called“complementarity determining regions” or CDRs. The CDRs from the twochains of each heavy chain/light chain pair mentioned above typicallyare aligned by the framework regions to form a structure that bindsspecifically with a specific epitope on the target protein (e.g.,PCSK9). From N-terminal to C-terminal, naturally-occurring light andheavy chain variable regions both typically conform with the followingorder of these elements: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. Anumbering system has been devised for assigning numbers to amino acidsthat occupy positions in each of these domains. This numbering system isdefined in Kabat Sequences of Proteins of Immunological Interest (1987and 1991, NIH, Bethesda, Md.), or Chothia & Lesk, 1987, J. Mol. Biol.196:901-917; Chothia et al., 1989, Nature 342:878-883.

Various heavy chain and light chain variable regions are provided hereinand are depicted in FIGS. 2A-3JJ and 3LL-3BBB. In some embodiments, eachof these variable regions can be attached to the above heavy and lightchain constant regions to form a complete antibody heavy and lightchain, respectively. Further, each of the so generated heavy and lightchain sequences can be combined to form a complete antibody structure.

Specific examples of some of the variable regions of the light and heavychains of the antibodies that are provided and their corresponding aminoacid sequences are summarized in TABLE 2.

TABLE 2 Exemplary Heavy and Light Chain Variable Regions Light/HeavyAntibody SEQ ID NO 30A4  5/74 3C4  7/85 23B5  9/71 25G4 10/72 31H4 12/6727B2 13/87 25A7 15/58 27H5 16/52 26H5 17/51 31D1 18/53 20D10 19/48 27E720/54 30B9 21/55 19H9 22/56 26E10 23/49 21B12 23/49 17C2 24/57 23G126/50 13H1 28/91 9C9 30/64 9H6 31/62 31A4 32/89 1A12 33/65 16F12 35/7922E2 36/80 27A6 37/76 28B12 38/77 28D6 39/78 31G11 40/81 13B5 42/6931B12 44/81 3B6 46/60

Again, each of the exemplary variable heavy chains listed in Table 2 canbe combined with any of the exemplary variable light chains shown inTable 2 to form an antibody. Table 2 shows exemplary light and heavychain pairings found in several of the antibodies disclosed herein. Insome instances, the antibodies include at least one variable heavy chainand one variable light chain from those listed in Table 2. In otherinstances, the antibodies contain two identical light chains and twoidentical heavy chains. As an example, an antibody or antigen bindingprotein can include a heavy chain and a light chain, two heavy chains,or two light chains. In some embodiments the antigen binding proteincomprises (and/or consists) of 1, 2, and/or 3 heavy and/or light CDRsfrom at least one of the sequences listed in Table 2 (CDRs for thesequences are outlined in FIGS. 2A-3D, and other embodiments in FIGS.3CCC-3JJJ and 15A-15D). In some embodiments, all 6 CDRs (CDR1-3 from thelight (CDRL1, CDRL2, CDRL3) and CDR1-3 from the heavy (CDRH1, CDRH2, andCDRH3)) are part of the ABP. In some embodiments, 1, 2, 3, 4, 5, or moreCDRs are included in the ABP. In some embodiments, one heavy and onelight CDR from the CDRs in the sequences in Table 2 is included in theABP (CDRs for the sequences in table 2 are outlined in FIGS. 2A-3D). Insome embodiments, additional sections (e.g., as depicted in FIG. 2A-2D,3A-3D, and other embodiments in 3CCC-3JJJ and 15A-15D) are also includedin the ABP. Examples of CDRs and FRs for the heavy and light chainsnoted in Table 2 are outlined in FIGS. 2A-3D (and other embodiments inFIGS. 3CCC-3JJJ and 15A-15D). Optional light chain variable sequences(including CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4) can be selectedfrom the following: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42, 44, and 46.Optional heavy chain variable sequences (including CDR1, CDR2, CDR3,FR1, FR2, FR3, and FR4) can be selected from the following: 74, 85, 71,72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89,65, 79, 80, 76, 77, 78, 83, 69, 81, and 60. In some of the entries inFIG. 2A-3D, variations of the sequences or alternative boundaries of theCDRs and FRs are identified. These alternatives are identified with a“v1” following the ABP name. As most of these alternatives are minor innature, only sections with differences are displayed in the table. It isunderstood that the remaining section of the light or heavy chain is thesame as shown for the base ABP in the other panels. Thus, for example,19H9v1 in FIG. 2C has the same FR1, CDR1, and FR2 as 19H9 in FIG. 2A asthe only difference is noted in FIG. 2C. For three of the nucleic acidsequences (ABPs 26E10, 30B9, and 31B12), additional alternative nucleicacid sequences are provided in the figures. As will be appreciated byone of skill in the art, no more than one such sequence need actually beused in the creation of an antibody or ABP. Indeed, in some embodiments,only one or neither of the specific heavy or light chain nucleic acidsneed be present.

In some embodiments, the ABP is encoded by a nucleic acid sequence thatcan encode any of the protein sequences in Table 2.

In some embodiments, the ABP binds selectively to the form of PCSK9 thatbinds to LDLR (e.g., the autocatalyzed form of the molecule). In someembodiments, the antigen binding protein does not bind to the c-terminusof the cataylytic domain (e.g., the 5. 5-10, 10-15, 15-20, 20-25, 25-30,30-40 most amino acids in the c-terminus). In some embodiments, theantigen binding protein does not bind to the n-terminus of the catalyticdomain (e.g., the 5. 5-10, 10-15, 15-20, 20-25, 25-30, 30-40 most aminoacids in the n-terminus). In some embodiments, the ABP binds to aminoacids within amino acids 1-100 of the mature form of PCSK9. In someembodiments, the ABP binds to amino acids within (and/or amino acidsequences consisting of) amino acids 31-100, 100-200, 31-152, 153-692,200-300, 300-400, 452-683, 400-500, 500-600, 31-692, 31-449, and/or600-692. In some embodiments, the ABP binds to the catalytic domain. Insome embodiments, the neutralizing and/or non-neutralizing ABP binds tothe prodomain. In some embodiments, the ABP binds to both the catalyticand pro domains. In some embodiments, the ABP binds to the catalyticdomain so as to obstruct an area on the catalytic domain that interactswith the pro domain. In some embodiments, the ABP binds to the catalyticdomain at a location or surface that the pro-domain interacts with asoutlined in Piper et al. (Structure 15:1-8 (2007), the entirety of whichis hereby incorporated by reference, including the structuralrepresentations therein). In some embodiments, the ABP binds to thecatalytic domain and restricts the mobility of the prodomain. In someembodiments, the ABP binds to the catalytic domain without binding tothe pro-domain. In some embodiments, the ABP binds to the catalyticdomain, without binding to the pro-domain, while preventing thepro-domain from reorienting to allow PCSK9 to bind to LDLR. In someembodiments, the ABP binds in the same epitope as those surroundingresidues 149-152 of the pro-domain in Piper et al. In some embodiments,the ABPs bind to the groove (as outlined in Piper et al.) on the Vdomain. In some embodiments, the ABPs bind to the histidine-rich patchproximal to the groove on the V domain. In some embodiments, suchantibodies (that bind to the V domain) are not neutralizing. In someembodiments, antibodies that bind to the V domain are neutralizing. Insome embodiments, the neutralizing ABPs prevent the binding of PCSK9 toLDLR. In some embodiments, the neturalizing ABPs, while preventing thePCSK9 degradation of LDLR, do not prevent the binding of PCSK9 to LDLR(for example ABP 31A4). In some embodiments, the ABP binds to or blocksat least one of the histidines depicted in Figure 4 of the Piper et al.paper. In some embodiments, the ABP blocks the catalytic triad in PCSK9.

In some embodiments, the antibody binds selectively to variant PCSK9proteins, e.g., D374Y over wild type PCSK9. In some embodiments, theseantibodies bind to the variant at least twice as strongly as the wildtype, and preferably 2-5, 5-10, 10-100, 100-1000, 1000-10,000 fold ormore to the mutant than the wild type (as measured via a K_(d)). In someembodiments, the antibody selectively inhibits variant D374Y PCSK9 frominteracting with LDLR over wild type PCSK9's ability to interact withLDLR. In some embodiments, these antibodies block the variant's abilityto bind to LDLR more strongly than the wild type's ability, e.g., atleast twice as strongly as the wild type, and preferably 2-5, 5-10,10-100, 100-1000 fold or more to the mutant than the wild type (asmeasured via an IC₅₀). In some embodiments, the antibody binds to andneutralizes both wild type PCSK9 and variant forms of PCSK9, such asD374Y at similar levels. In some embodiments, the antibody binds toPCSK9 to prevent variants of LDLR from binding to PCSK9. In someembodiments, the variants of LDLR are at least 50% identical to humanLDLR. It is noted that variants of LDLR are known to those of skill inthe art (e.g., Brown M S et al, “Calcium cages, acid baths and recyclingreceptors” Nature 388: 629-630, 1997). In some embodiments, the ABP canraise the level of effective LDLR in heterozygote familialhypercholesterolemia (where a loss-of function variant of LDLR ispresent).

In some embodiments, the ABP binds to (but does not block) variants ofPCSK9 that are at least 50%, 50-60, 60-70, 70-80, 80-90, 90-95, 95-99,or greater percent identity to the form of PCSK9 depicted in FIG. 1Aand/or FIG. 1B. In some embodiments, the ABP binds to (but does notblock) variants of PCSK9 that are at least 50%, 50-60, 60-70, 70-80,80-90, 90-95, 95-99, or greater percent identity to the mature form ofPCSK9 depicted in FIG. 1A and/or FIG. 1B. In some embodiments, the ABPbinds to and prevents variants of PCSK9 that are at least 50%, 50-60,60-70, 70-80, 80-90, 90-95, 95-99, or greater percent identity to theform of PCSK9 depicted in FIG. 1A and/or FIG. 1B from interacting withLDLR. In some embodiments, the ABP binds to and prevents variants ofPCSK9 that are at least 50, 50-60, 60-70, 70-80, 80-90, 90-95, 95-99, orgreater percent identity to the mature form of PCSK9 depicted in FIG. 1Bfrom interacting with LDLR. In some embodiments, the variant of PCSK9 isa human variant, such as variants at position 474, E620G, and/or E670G.In some embodiments, the amino acid at position 474 is valine (as inother humans) or threonine (as in cyno and mouse). Given thecross-reactivity data presented herein, it is believed that the presentantibodies will readily bind to the above variants.

In some embodiments, the ABP binds to an epitope bound by one of theantibodies described in Table 2. In some embodiments, the antigenbinding proteins bind to a specific conformational state of PCSK9 so asto prevent PCSK9 from interacting with LDLR.

Humanized Antigen Binding Proteins (e.g., Antibodies)

As described herein, an antigen binding protein to PCSK9 can comprise ahumanized antibody and/or part thereof. An important practicalapplication of such a strategy is the “humanization” of the mousehumoral immune system.

In certain embodiments, a humanized antibody is substantiallynon-immunogenic in humans. In certain embodiments, a humanized antibodyhas substantially the same affinity for a target as an antibody fromanother species from which the humanized antibody is derived. See, e.g.,U.S. Pat. No. 5,530,101, U.S. Pat. No. 5,693,761; U.S. Pat. No.5,693,762; U.S. Pat. No. 5,585,089.

In certain embodiments, amino acids of an antibody variable domain thatcan be modified without diminishing the native affinity of the antigenbinding domain while reducing its immunogenicity are identified. See,e.g., U.S. Pat. Nos. 5,766,886 and 5,869,619.

In certain embodiments, modification of an antibody by methods known inthe art is typically designed to achieve increased binding affinity fora target and/or to reduce immunogenicity of the antibody in therecipient. In certain embodiments, humanized antibodies are modified toeliminate glycosylation sites in order to increase affinity of theantibody for its cognate antigen. See, e.g., Co et al., Mol. Immunol.,30:1361-1367 (1993). In certain embodiments, techniques such as“reshaping,” “hyperchimerization,” or “veneering/resurfacing” are usedto produce humanized antibodies. See, e.g., Vaswami et al., Annals ofAllergy, Asthma, & Immunol. 81:105 (1998); Roguska et al., Prot.Engineer., 9:895-904 (1996); and U.S. Pat. No. 6,072,035. In certainsuch embodiments, such techniques typically reduce antibodyimmunogenicity by reducing the number of foreign residues, but do notprevent anti-idiotypic and anti-allotypic responses following repeatedadministration of the antibodies. Certain other methods for reducingimmunogenicity are described, e.g., in Gilliland et al., J. Immunol.,62(6): 3663-71 (1999).

In certain instances, humanizing antibodies results in a loss of antigenbinding capacity. In certain embodiments, humanized antibodies are “backmutated.” In certain such embodiments, the humanized antibody is mutatedto include one or more of the amino acid residues found in the donorantibody. See, e.g., Saldanha et al., Mol Immunol 36:709-19 (1999).

In certain embodiments the complementarity determining regions (CDRs) ofthe light and heavy chain variable regions of an antibody to PCSK9 canbe grafted to framework regions (FRs) from the same, or another,species. In certain embodiments, the CDRs of the light and heavy chainvariable regions of an antibody to PCSK9 can be grafted to consensushuman FRs. To create consensus human FRs, in certain embodiments, FRsfrom several human heavy chain or light chain amino acid sequences arealigned to identify a consensus amino acid sequence. In certainembodiments, the FRs of an antibody to PCSK9 heavy chain or light chainare replaced with the FRs from a different heavy chain or light chain.In certain embodiments, rare amino acids in the FRs of the heavy andlight chains of an antibody to PCSK9 are not replaced, while the rest ofthe FR amino acids are replaced. Rare amino acids are specific aminoacids that are in positions in which they are not usually found in FRs.In certain embodiments, the grafted variable regions from an antibody toPCSK9 can be used with a constant region that is different from theconstant region of an antibody to PCSK9. In certain embodiments, thegrafted variable regions are part of a single chain Fv antibody. CDRgrafting is described, e.g., in U.S. Pat. Nos. 6,180,370, 6,054,297,5,693,762, 5,859,205, 5,693,761, 5,565,332, 5,585,089, and 5,530,101,and in Jones et al., Nature, 321: 522-525 (1986); Riechmann et al.,Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239:1534-1536(1988), Winter, FEBS Letts., 430:92-94 (1998), which are herebyincorporated by reference for any purpose.

Human Antigen Binding Proteins (e.g., Antibodies)

As described herein, an antigen binding protein that binds to PCSK9 cancomprise a human (i.e., fully human) antibody and/or part thereof. Incertain embodiments, nucleotide sequences encoding, and amino acidsequences comprising, heavy and light chain immunoglobulin molecules,particularly sequences corresponding to the variable regions areprovided. In certain embodiments, sequences corresponding tocomplementarity determining regions (CDR's), specifically from CDR1through CDR3, are provided. According to certain embodiments, ahybridoma cell line expressing such an immunoglobulin molecule isprovided. According to certain embodiments, a hybridoma cell lineexpressing such a monoclonal antibody is provided. In certainembodiments a hybridoma cell line is selected from at least one of thecell lines described in Table 2, e.g., 21B12, 16F12 and 31H4. In certainembodiments, a purified human monoclonal antibody to human PCSK9 isprovided.

One can engineer mouse strains deficient in mouse antibody productionwith large fragments of the human Ig loci in anticipation that such micewould produce human antibodies in the absence of mouse antibodies. Largehuman Ig fragments can preserve the large variable gene diversity aswell as the proper regulation of antibody production and expression. Byexploiting the mouse machinery for antibody diversification andselection and the lack of immunological tolerance to human proteins, thereproduced human antibody repertoire in these mouse strains can yieldhigh affinity fully human antibodies against any antigen of interest,including human antigens. Using the hybridoma technology,antigen-specific human MAbs with the desired specificity can be producedand selected. Certain exemplary methods are described in WO 98/24893,U.S. Pat. No. 5,545,807, EP 546073, and EP 546073.

In certain embodiments, one can use constant regions from species otherthan human along with the human variable region(s).

The ability to clone and reconstruct megabase sized human loci in yeastartificial chromosomes (YACs) and to introduce them into the mousegermline provides an approach to elucidating the functional componentsof very large or crudely mapped loci as well as generating useful modelsof human disease. Furthermore, the utilization of such technology forsubstitution of mouse loci with their human equivalents could provideinsights into the expression and regulation of human gene productsduring development, their communication with other systems, and theirinvolvement in disease induction and progression.

Human antibodies avoid some of the problems associated with antibodiesthat possess murine or rat variable and/or constant regions. Thepresence of such murine or rat derived proteins can lead to the rapidclearance of the antibodies or can lead to the generation of an immuneresponse against the antibody by a patient. In order to avoid theutilization of murine or rat derived antibodies, fully human antibodiescan be generated through the introduction of functional human antibodyloci into a rodent, other mammal or animal so that the rodent, othermammal or animal produces fully human antibodies.

Humanized antibodies are those antibodies that, while initially startingoff containing antibody amino acid sequences that are not human, havehad at least some of these nonhuman antibody amino acid sequencesreplaced with human antibody sequences. This is in contrast with humanantibodies, in which the antibody is encoded (or capable of beingencoded) by genes possessed a human.

Antigen Binding Protein Variants

Other antibodies that are provided are variants of the ABPs listed aboveformed by combination or subparts of the variable heavy and variablelight chains shown in Table 2 and comprise variable light and/orvariable heavy chains that each have at least 50%, 50-60, 60-70, 70-80%,80-85%, 85-90%, 90-95%, 95-97%, 97-99%, or above 99% identity to theamino acid sequences of the sequences in Table 2 (either the entiresequence or a subpart of the sequence, e.g., one or more CDR). In someinstances, such antibodies include at least one heavy chain and onelight chain, whereas in other instances the variant forms contain twoidentical light chains and two identical heavy chains (or subpartsthereof). In some embodiments, the sequence comparison in FIG. 2A-3D(and 13A-13J and other embodiments in 15A-15D) can be used in order toidentify sections of the antibodies that can be modified by observingthose variations that impact binding and those variations that do notappear to impact binding. For example, by comparing similar sequences,one can identify those sections (e.g., particular amino acids) that canbe modified and how they can be modified while still retaining (orimproving) the functionality of the ABP. In some embodiments, variantsof ABPs include those consensus groups and sequences depicted in FIGS.13A, 13C, 13F, 13G, 13H, 13I and/or 13J and variations are allowed inthe positions identified as variable in the figures. The CDRs shown inFIGS. 13A, 13C, 13F, and 13G were defined based upon a hybridcombination of the Chothia method (based on the location of thestructural loop regions, see, e.g., “Standard conformations for thecanonical structures of immunoglobulins,” Bissan Al-Lazikani, Arthur M.Lesk and Cyrus Chothia, Journal of Molecular Biology, 273(4): 927-948, 7Nov. (1997)) and the Kabat method (based on sequence variability, see,e.g., Sequences of Proteins of Immunological Interest, Fifth Edition.NIH Publication No. 91-3242, Kabat et al., (1991)). Each residuedetermined by either method, was included in the final list of CDRresidues (and is presented in FIGS. 13A, 13C, 13F, and 13G). The CDRs inFIGS. 13H, 13I, and 13J were obtained by the Kabat method alone. Unlessspecified otherwise, the defined consensus sequences, CDRs, and FRs inFIGS. 13H-13J will define and control the noted CDRs and FRs for thereferenced ABPs in FIG. 13.

In certain embodiments, an antigen binding protein comprises a heavychain comprising a variable region comprising an amino acid sequence atleast 90% identical to an amino acid sequence selected from at least oneof the sequences of SEQ ID NO: 74, 85, 71, 72, 67, 87, 58, 52, 51, 53,48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80, 76, 77, 78, 83,69, 81, and 60. In certain embodiments, an antigen binding proteincomprises a heavy chain comprising a variable region comprising an aminoacid sequence at least 95% identical to an amino acid sequence selectedfrom at least one of the sequences of SEQ ID NO: 74, 85, 71, 72, 67, 87,58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65, 79, 80,76, 77, 78, 83, 69, 81, and 60. In certain embodiments, an antigenbinding protein comprises a heavy chain comprising a variable regioncomprising an amino acid sequence at least 99% identical to an aminoacid sequence selected from at least one of the sequences of SEQ ID NO:74, 85, 71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91,64, 62, 89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60.

In some embodiments, the antigen binding protein comprises a sequencethat is at least 90%, 90-95%, and/or 95-99% identical to one or moreCDRs from the CDRs in at least one of sequences of SEQ ID NO: 74, 85,71, 72, 67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62,89, 65, 79, 80, 76, 77, 78, 83, 69, 81, and 60. In some embodiments, 1,2, 3, 4, 5, or 6 CDR (each being at least 90%, 90-95%, and/or 95-99%identical to the above sequences) is present.

In some embodiments, the antigen binding protein comprises a sequencethat is at least 90%, 90-95%, and/or 95-99% identical to one or more FRsfrom the FRs in at least one of sequences of SEQ ID NO: 74, 85, 71, 72,67, 87, 58, 52, 51, 53, 48, 54, 55, 56, 49, 57, 50, 91, 64, 62, 89, 65,79, 80, 76, 77, 78, 83, 69, 81, and 60. In some embodiments, 1, 2, 3, or4 FR (each being at least 90%, 90-95%, and/or 95-99% identical to theabove sequences) is present.

In certain embodiments, an antigen binding protein comprises a lightchain comprising a variable region comprising an amino acid sequence atleast 90% identical to an amino acid sequence selected from at least oneof the sequences of SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38, 39, 40, 42,44, and 46. In certain embodiments, an antigen binding protein comprisesa light chain comprising a variable region comprising an amino acidsequence at least 95% identical to an amino acid sequence selected fromat least one of the sequences of SEQ ID NO: 5, 7, 9, 10, 12, 13, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35, 36, 37, 38,39, 40, 42, 44, and 46. In certain embodiments, an antigen bindingprotein comprises a light chain comprising a variable region comprisingan amino acid sequence at least 99% identical to an amino acid sequenceselected from at least one of the sequences of SEQ ID NO: 5, 7, 9, 10,12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33,35, 36, 37, 38, 39, 40, 42, 44, and 46.

In some embodiments, the antigen binding protein comprises a sequencethat is at least 90%, 90-95%, and/or 95-99% identical to one or moreCDRs from the CDRs in at least one of sequences of SEQ ID NO: 5, 7, 9,10, 12, 13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32,33, 35, 36, 37, 38, 39, 40, 42, 44, and 46. In some embodiments, 1, 2,3, 4, 5, or 6 CDR (each being at least 90%, 90-95%, and/or 95-99%identical to the above sequences) is present.

In some embodiments, the antigen binding protein comprises a sequencethat is at least 90%, 90-95%, and/or 95-99% identical to one or more FRsfrom the FRs in at least one of sequences of SEQ ID NO: 5, 7, 9, 10, 12,13, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 28, 30, 31, 32, 33, 35,36, 37, 38, 39, 40, 42, 44, and 46. In some embodiments, 1, 2, 3, or 4FR (each being at least 90%, 90-95%, and/or 95-99% identical to theabove sequences) is present.

In light of the present disclosure, a skilled artisan will be able todetermine suitable variants of the ABPs as set forth herein usingwell-known techniques. In certain embodiments, one skilled in the artcan identify suitable areas of the molecule that may be changed withoutdestroying activity by targeting regions not believed to be importantfor activity. In certain embodiments, one can identify residues andportions of the molecules that are conserved among similar polypeptides.In certain embodiments, even areas that can be important for biologicalactivity or for structure can be subject to conservative amino acidsubstitutions without destroying the biological activity or withoutadversely affecting the polypeptide structure.

Additionally, one skilled in the art can review structure-functionstudies identifying residues in similar polypeptides that are importantfor activity or structure. In view of such a comparison, one can predictthe importance of amino acid residues in a protein that correspond toamino acid residues which are important for activity or structure insimilar proteins. One skilled in the art can opt for chemically similaramino acid substitutions for such predicted important amino acidresidues.

One skilled in the art can also analyze the three-dimensional structureand amino acid sequence in relation to that structure in similar ABPs.In view of such information, one skilled in the art can predict thealignment of amino acid residues of an antibody with respect to itsthree dimensional structure. In certain embodiments, one skilled in theart can choose not to make radical changes to amino acid residuespredicted to be on the surface of the protein, since such residues canbe involved in important interactions with other molecules. Moreover,one skilled in the art can generate test variants containing a singleamino acid substitution at each desired amino acid residue. The variantscan then be screened using activity assays known to those skilled in theart. Such variants can be used to gather information about suitablevariants. For example, if one discovered that a change to a particularamino acid residue resulted in destroyed, undesirably reduced, orunsuitable activity, variants with such a change can be avoided. Inother words, based on information gathered from such routineexperiments, one skilled in the art can readily determine the aminoacids where further substitutions should be avoided either alone or incombination with other mutations.

A number of scientific publications have been devoted to the predictionof secondary structure. See Moult J., Curr. Op. in Biotech.,7(4):422-427 (1996), Chou et al., Biochemistry, 13(2):222-245 (1974);Chou et al., Biochemistry, 113(2):211-222 (1974); Chou et al., Adv.Enzymol. Relat. Areas Mol. Biol., 47:45-148 (1978); Chou et al., Ann.Rev. Biochem., 47:251-276 and Chou et al., Biophys. J., 26:367-384(1979). Moreover, computer programs are currently available to assistwith predicting secondary structure. One method of predicting secondarystructure is based upon homology modeling. For example, two polypeptidesor proteins which have a sequence identity of greater than 30%, orsimilarity greater than 40% often have similar structural topologies.The recent growth of the protein structural database (PDB) has providedenhanced predictability of secondary structure, including the potentialnumber of folds within a polypeptide's or protein's structure. See Holmet al., Nucl. Acid. Res., 27(1):244-247 (1999). It has been suggested(Brenner et al., Curr. Op. Struct. Biol., 7(3):369-376 (1997)) thatthere are a limited number of folds in a given polypeptide or proteinand that once a critical number of structures have been resolved,structural prediction will become dramatically more accurate.

Additional methods of predicting secondary structure include “threading”(Jones, D., Curr. Opin. Struct. Biol., 7(3):377-87 (1997); Sippl et al.,Structure, 4(1):15-19 (1996)), “profile analysis” (Bowie et al.,Science, 253:164-170 (1991); Gribskov et al., Meth. Enzym., 183:146-159(1990); Gribskov et al., Proc. Nat. Acad. Sci. USA, 84(13):4355-4358(1987)), and “evolutionary linkage” (See Holm, supra (1999), andBrenner, supra (1997)).

In certain embodiments, antigen binding protein variants includeglycosylation variants wherein the number and/or type of glycosylationsite has been altered compared to the amino acid sequences of a parentpolypeptide. In certain embodiments, protein variants comprise a greateror a lesser number of N-linked glycosylation sites than the nativeprotein. An N-linked glycosylation site is characterized by thesequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residuedesignated as X can be any amino acid residue except proline. Thesubstitution of amino acid residues to create this sequence provides apotential new site for the addition of an N-linked carbohydrate chain.Alternatively, substitutions which eliminate this sequence will removean existing N-linked carbohydrate chain. Also provided is arearrangement of N-linked carbohydrate chains wherein one or moreN-linked glycosylation sites (typically those that are naturallyoccurring) are eliminated and one or more new N-linked sites arecreated. Additional preferred antibody variants include cysteinevariants wherein one or more cysteine residues are deleted from orsubstituted for another amino acid (e.g., serine) as compared to theparent amino acid sequence. Cysteine variants can be useful whenantibodies must be refolded into a biologically active conformation suchas after the isolation of insoluble inclusion bodies. Cysteine variantsgenerally have fewer cysteine residues than the native protein, andtypically have an even number to minimize interactions resulting fromunpaired cysteines.

According to certain embodiments, amino acid substitutions are thosewhich: (1) reduce susceptibility to proteolysis, (2) reducesusceptibility to oxidation, (3) alter binding affinity for formingprotein complexes, (4) alter binding affinities, and/or (4) confer ormodify other physiocochemical or functional properties on suchpolypeptides. According to certain embodiments, single or multiple aminoacid substitutions (in certain embodiments, conservative amino acidsubstitutions) can be made in the naturally-occurring sequence (incertain embodiments, in the portion of the polypeptide outside thedomain(s) forming intermolecular contacts). In certain embodiments, aconservative amino acid substitution typically may not substantiallychange the structural characteristics of the parent sequence (e.g., areplacement amino acid should not tend to break a helix that occurs inthe parent sequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence). Examples of art-recognizedpolypeptide secondary and tertiary structures are described in Proteins,Structures and Molecular Principles (Creighton, Ed., W.H. Freeman andCompany, New York (1984)); Introduction to Protein Structure (C. Branden& J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); andThornton et al., Nature, 354:105 (1991), which are each incorporatedherein by reference.

In some embodiments, the variants are variants of the nucleic acidsequences of the ABPs disclosed herein. One of skill in the art willappreciate that the above discussion can be used for identifying,evaluating, and/creating ABP protein variants and also for nucleic acidsequences that can encode for those protein variants. Thus, nucleic acidsequences encoding for those protein variants (as well as nucleic acidsequences that encode for the ABPs in Table 2, but are different fromthose explicitly disclosed herein) are contemplated. For example, an ABPvariant can have at least 80, 80-85, 85-90, 90-95, 95-97, 97-99 orgreater identity to at least one nucleic acid sequence described in SEQID NOs: 152, 153, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,146, 147, 148, 149, 150, 151 or at least one to six (and variouscombinations thereof) of the CDR(s) encoded by the nucleic acidsequences in SEQ ID NOs: 152, 153, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114,115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128,129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142,143, 144, 145, 146, 147, 148, 149, 150, and 151.

In some embodiments, the antibody (or nucleic acid sequence encoding it)is a variant if the nucleic acid sequence that encodes the particularABP (or the nucleic acid sequence itself) can selectively hybridize toany of the nucleic acid sequences that encode the proteins in Table 2(such as, but not limited to SEQ ID NO: 152, 153, 92, 93, 94, 95, 96,97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111,112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, and 151) understringent conditions. In one embodiment, suitable moderately stringentconditions include prewashing in a solution of 5×SSC; 0.5% SDS, 1.0 mMEDTA (pH 8:0); hybridizing at 50° C., −65° C., 5×SSC, overnight or, inthe event of cross-species homology, at 45° C. with 0.5×SSC; followed bywashing twice at 65° C. for 20 minutes with each of 2×, 0.5× and 0.2×SSCcontaining 0.1% SDS. Such hybridizing DNA sequences are also within thescope of this invention, as are nucleotide sequences that, due to codedegeneracy, encode an antibody polypeptide that is encoded by ahybridizing DNA sequence and the amino acid sequences that are encodedby these nucleic acid sequences. In some embodiments, variants of CDRsinclude nucleic acid sequences and the amino acid sequences encoded bythose sequences, that hybridize to one or more of the CDRs within thesequences noted above (individual CDRs can readily be determined inlight of FIGS. 2A-3D, and other embodiments in FIGS. 3CCC-3JJJ and15A-15D). The phrase “selectively hybridize” referred to in this contextmeans to detectably and selectively bind. Polynucleotides,oligonucleotides and fragments thereof in accordance with the inventionselectively hybridize to nucleic acid strands under hybridization andwash conditions that minimize appreciable amounts of detectable bindingto nonspecific nucleic acids. High stringency conditions can be used toachieve selective hybridization conditions as known in the art anddiscussed herein. Generally, the nucleic acid sequence homology betweenthe polynucleotides, oligonucleotides, and fragments of the inventionand a nucleic acid sequence of interest will be at least 80%, and moretypically with preferably increasing homologies of at least 85%, 90%,95%, 99%, and 100%. Two amino acid sequences are homologous if there isa partial or complete identity between their sequences. For example, 85%homology means that 85% of the amino acids are identical when the twosequences are aligned for maximum matching. Gaps (in either of the twosequences being matched) are allowed in maximizing matching; gap lengthsof 5 or less are preferred with 2 or less being more preferred.Alternatively and preferably, two protein sequences (or polypeptidesequences derived from them of at least 30 amino acids in length) arehomologous, as this term is used herein, if they have an alignment scoreof at more than 5 (in standard deviation units) using the program ALIGNwith the mutation data matrix and a gap penalty of 6 or greater. SeeDayhoff, M. O., in Atlas of Protein Sequence and Structure, pp. 101-110(Volume 5, National Biomedical Research Foundation (1972)) andSupplement 2 to this volume, pp. 1-10. The two sequences or partsthereof are more preferably homologous if their amino acids are greaterthan or equal to 50% identical when optimally aligned using the ALIGNprogram. The term “corresponds to” is used herein to mean that apolynucleotide sequence is homologous (i.e., is identical, not strictlyevolutionarily related) to all or a portion of a referencepolynucleotide sequence, or that a polypeptide sequence is identical toa reference polypeptide sequence. In contradistinction, the term“complementary to” is used herein to mean that the complementarysequence is homologous to all or a portion of a reference polynucleotidesequence. For illustration, the nucleotide sequence “TATAC” correspondsto a reference sequence “TATAC” and is complementary to a referencesequence “GTATA”.

Preparation of Antigen Binding Proteins (e.g., Antibodies)

In certain embodiments, antigen binding proteins (such as antibodies)are produced by immunization with an antigen (e.g., PCSK9). In certainembodiments, antibodies can be produced by immunization with full-lengthPCSK9, a soluble form of PCSK9, the catalytic domain alone, the matureform of PCSK9 shown in FIG. 1A, a splice variant form of PCSK9, or afragment thereof. In certain embodiments, the antibodies of theinvention can be polyclonal or monoclonal, and/or can be recombinantantibodies. In certain embodiments, antibodies of the invention arehuman antibodies prepared, for example, by immunization of transgenicanimals capable of producing human antibodies (see, for example, PCTPublished Application No. WO 93/12227).

In certain embodiments, certain strategies can be employed to manipulateinherent properties of an antibody, such as the affinity of an antibodyfor its target. Such strategies include, but are not limited to, the useof site-specific or random mutagenesis of the polynucleotide moleculeencoding an antibody to generate an antibody variant. In certainembodiments, such generation is followed by screening for antibodyvariants that exhibit the desired change, e.g. increased or decreasedaffinity.

In certain embodiments, the amino acid residues targeted in mutagenicstrategies are those in the CDRs. In certain embodiments, amino acids inthe framework regions of the variable domains are targeted. In certainembodiments, such framework regions have been shown to contribute to thetarget binding properties of certain antibodies. See, e.g., Hudson,Curr. Opin. Biotech., 9:395-402 (1999) and references therein.

In certain embodiments, smaller and more effectively screened librariesof antibody variants are produced by restricting random or site-directedmutagenesis to hyper-mutation sites in the CDRs, which are sites thatcorrespond to areas prone to mutation during the somatic affinitymaturation process. See, e.g., Chowdhury & Pastan, Nature Biotech., 17:568-572 (1999) and references therein. In certain embodiments, certaintypes of DNA elements can be used to identify hyper-mutation sitesincluding, but not limited to, certain direct and inverted repeats,certain consensus sequences, certain secondary structures, and certainpalindromes. For example, such DNA elements that can be used to identifyhyper-mutation sites include, but are not limited to, a tetrabasesequence comprising a purine (A or G), followed by guainine (G),followed by a pyrimidine (C or T), followed by either adenosine orthymidine (A or T) (i.e., A/G-G-C/T-A/T). Another example of a DNAelement that can be used to identify hyper-mutation sites is the serinecodon, A-G-C/T.

Preparation of Fully Human ABPs (e.g., Antibodies)

In certain embodiments, a phage display technique is used to generatemonoclonal antibodies. In certain embodiments, such techniques producefully human monoclonal antibodies. In certain embodiments, apolynucleotide encoding a single Fab or Fv antibody fragment isexpressed on the surface of a phage particle. See, e.g., Hoogenboom etal., J. Mol. Biol., 227: 381 (1991); Marks et al., J Mol Biol 222: 581(1991); U.S. Pat. No. 5,885,793. In certain embodiments, phage are“screened” to identify those antibody fragments having affinity fortarget. Thus, certain such processes mimic immune selection through thedisplay of antibody fragment repertoires on the surface of filamentousbacteriophage, and subsequent selection of phage by their binding totarget. In certain such procedures, high affinity functionalneutralizing antibody fragments are isolated. In certain suchembodiments (discussed in more detail below), a complete repertoire ofhuman antibody genes is created by cloning naturally rearranged human Vgenes from peripheral blood lymphocytes. See, e.g., Mullinax et al.,Proc Natl Acad Sci (USA), 87: 8095-8099 (1990).

According to certain embodiments, antibodies of the invention areprepared through the utilization of a transgenic mouse that has asubstantial portion of the human antibody producing genome inserted butthat is rendered deficient in the production of endogenous, murineantibodies. Such mice, then, are capable of producing humanimmunoglobulin molecules and antibodies and are deficient in theproduction of murine immunoglobulin molecules and antibodies.Technologies utilized for achieving this result are disclosed in thepatents, applications and references disclosed in the specification,herein. In certain embodiments, one can employ methods such as thosedisclosed in PCT Published Application No. WO 98/24893 or in Mendez etal., Nature Genetics, 15:146-156 (1997), which are hereby incorporatedby reference for any purpose.

Generally, fully human monoclonal ABPs (e.g., antibodies) specific forPCSK9 can be produced as follows. Transgenic mice containing humanimmunoglobulin genes are immunized with the antigen of interest, e.g.PCSK9, lymphatic cells (such as B-cells) from the mice that expressantibodies are obtained. Such recovered cells are fused with amyeloid-type cell line to prepare immortal hybridoma cell lines, andsuch hybridoma cell lines are screened and selected to identifyhybridoma cell lines that produce antibodies specific to the antigen ofinterest. In certain embodiments, the production of a hybridoma cellline that produces antibodies specific to PCSK9 is provided.

In certain embodiments, fully human antibodies are produced by exposinghuman splenocytes (B or T cells) to an antigen in vitro, and thenreconstituting the exposed cells in an immunocompromised mouse, e.g.SCID or nod/SCID. See, e.g., Brams et al., J. Immunol. 160: 2051-2058(1998); Carballido et al., Nat. Med., 6: 103-106 (2000). In certain suchapproaches, engraftment of human fetal tissue into SCID mice (SCID-hu)results in long-term hematopoiesis and human T-cell development. See,e.g., McCune et al., Science, 241:1532-1639 (1988); Ifversen et al.,Sem. Immunol., 8:243-248 (1996). In certain instances, humoral immuneresponse in such chimeric mice is dependent on co-development of humanT-cells in the animals. See, e.g., Martensson et al., Immunol.,83:1271-179 (1994). In certain approaches, human peripheral bloodlymphocytes are transplanted into SCID mice. See, e.g., Mosier et al.,Nature, 335:256-259 (1988). In certain such embodiments, when suchtransplanted cells are treated either with a priming agent, such asStaphylococcal Enterotoxin A (SEA), or with anti-human CD40 monoclonalantibodies, higher levels of B cell production is detected. See, e.g.,Martensson et al., Immunol., 84: 224-230 (1995); Murphy et al., Blood,86:1946-1953 (1995).

Thus, in certain embodiments, fully human antibodies can be produced bythe expression of recombinant DNA in host cells or by expression inhybridoma cells. In other embodiments, antibodies can be produced usingthe phage display techniques described herein.

The antibodies described herein were prepared through the utilization ofthe XenoMouse® technology, as described herein. Such mice, then, arecapable of producing human immunoglobulin molecules and antibodies andare deficient in the production of murine immunoglobulin molecules andantibodies. Technologies utilized for achieving the same are disclosedin the patents, applications, and references disclosed in the backgroundsection herein. In particular, however, a preferred embodiment oftransgenic production of mice and antibodies therefrom is disclosed inU.S. patent application Ser. No. 08/759,620, filed Dec. 3, 1996 andInternational Patent Application Nos. WO 98/24893, published Jun. 11,1998 and WO 00/76310, published Dec. 21, 2000, the disclosures of whichare hereby incorporated by reference. See also Mendez et al., NatureGenetics, 15:146-156 (1997), the disclosure of which is herebyincorporated by reference.

Through the use of such technology, fully human monoclonal antibodies toa variety of antigens have been produced. Essentially, XenoMouse® linesof mice are immunized with an antigen of interest (e.g. PCSK9),lymphatic cells (such as B-cells) are recovered from the hyper-immunizedmice, and the recovered lymphocytes are fused with a myeloid-type cellline to prepare immortal hybridoma cell lines. These hybridoma celllines are screened and selected to identify hybridoma cell lines thatproduced antibodies specific to the antigen of interest. Provided hereinare methods for the production of multiple hybridoma cell lines thatproduce antibodies specific to PCSK9 Further, provided herein arecharacterization of the antibodies produced by such cell lines,including nucleotide and amino acid sequence analyses of the heavy andlight chains of such antibodies.

The production of the XenoMouse® strains of mice is further discussedand delineated in U.S. patent application Ser. No. 07/466,008, filedJan. 12, 1990, 07/610,515, filed Nov. 8, 1990, 07/919,297, filed Jul.24, 1992, 07/922,649, filed Jul. 30, 1992, 08/031,801, filed Mar. 15,1993, 08/112,848, filed Aug. 27, 1993, 08/234,145, filed Apr. 28, 1994,08/376,279, filed Jan. 20, 1995, 08/430, 938, filed Apr. 27, 1995,08/464,584, filed Jun. 5, 1995, 08/464,582, filed Jun. 5, 1995,08/463,191, filed Jun. 5, 1995, 08/462,837, filed Jun. 5, 1995,08/486,853, filed Jun. 5, 1995, 08/486,857, filed Jun. 5, 1995,08/486,859, filed Jun. 5, 1995, 08/462,513, filed Jun. 5, 1995,08/724,752, filed Oct. 2, 1996, 08/759,620, filed Dec. 3, 1996, U.S.Publication 2003/0093820, filed Nov. 30, 2001 and U.S. Pat. Nos.6,162,963, 6,150,584, 6,114,598, 6,075,181, and 5,939,598 and JapanesePatent Nos. 3 068 180 B2, 3 068 506 B2, and 3 068 507 B2. See alsoEuropean Patent No., EP 0 463 151 B1, grant published Jun. 12, 1996,International Patent Application No., WO 94/02602, published Feb. 3,1994, International Patent Application No., WO 96/34096, published Oct.31, 1996, WO 98/24893, published Jun. 11, 1998, WO 00/76310, publishedDec. 21, 2000. The disclosures of each of the above-cited patents,applications, and references are hereby incorporated by reference intheir entirety.

In an alternative approach, others, including GenPharm International,Inc., have utilized a “minilocus” approach. In the minilocus approach,an exogenous Ig locus is mimicked through the inclusion of pieces(individual genes) from the Ig locus. Thus, one or more V_(H) genes, oneor more D_(H) genes, one or more J_(H) genes, a mu constant region, andusually a second constant region (preferably a gamma constant region)are formed into a construct for insertion into an animal. This approachis described in U.S. Pat. No. 5,545,807 to Surani et al. and U.S. Pat.Nos. 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429,5,789,650, 5,814,318, 5,877,397, 5,874,299, and 6,255,458 each toLonberg & Kay, U.S. Pat. Nos. 5,591,669 and 6,023.010 to Krimpenfort &Berns, U.S. Pat. Nos. 5,612,205, 5,721,367, and 5,789,215 to Berns etal., and U.S. Pat. No. 5,643,763 to Choi & Dunn, and GenPharmInternational U.S. patent application Ser. No. 07/574,748, filed Aug.29, 1990, 07/575,962, filed Aug. 31, 1990, 07/810,279, filed Dec. 17,1991, 07/853,408, filed Mar. 18, 1992, 07/904,068, filed Jun. 23, 1992,07/990,860, filed Dec. 16, 1992, 08/053,131, filed Apr. 26, 1993,08/096,762, filed Jul. 22, 1993, 08/155,301, filed Nov. 18, 1993,08/161,739, filed Dec. 3, 1993, 08/165,699, filed Dec. 10, 1993,08/209,741, filed Mar. 9, 1994, the disclosures of which are herebyincorporated by reference. See also European Patent No. 0 546 073 B1,International Patent Application Nos. WO 92/03918, WO 92/22645, WO92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO96/14436, WO 97/13852, and WO 98/24884 and U.S. Pat. No. 5,981,175, thedisclosures of which are hereby incorporated by reference in theirentirety. See further Taylor et al., 1992, Chen et al., 1993, Tuaillonet al., 1993, Choi et al., 1993, Lonberg et al., (1994), Taylor et al.,(1994), and Tuaillon et al., (1995), Fishwild et al., (1996), thedisclosures of which are hereby incorporated by reference in theirentirety.

Kirin has also demonstrated the generation of human antibodies from micein which, through microcell fusion, large pieces of chromosomes, orentire chromosomes, have been introduced. See European PatentApplication Nos. 773 288 and 843 961, the disclosures of which arehereby incorporated by reference. Additionally, KM™ mice, which are theresult of cross-breeding of Kirin's Tc mice with Medarex's minilocus(Humab) mice have been generated. These mice possess the human IgHtranschromosome of the Kirin mice and the kappa chain transgene of theGenpharm mice (Ishida et al., Cloning Stem Cells, (2002) 4:91-102).

Human antibodies can also be derived by in vitro methods. Suitableexamples include but are not limited to phage display (CAT, Morphosys,Dyax, Biosite/Medarex, Xoma, Symphogen, Alexion (formerly Proliferon),Affimed) ribosome display (CAT), yeast display, and the like.

In some embodiments, the antibodies described herein possess human IgG4heavy chains as well as IgG2 heavy chains. Antibodies can also be ofother human isotypes, including IgG1. The antibodies possessed highaffinities, typically possessing a Kd of from about 10⁻⁶ through about10⁻¹³ M or below, when measured by various techniques.

As will be appreciated, antibodies can be expressed in cell lines otherthan hybridoma cell lines. Sequences encoding particular antibodies canbe used to transform a suitable mammalian host cell. Transformation canbe by any known method for introducing polynucleotides into a host cell,including, for example packaging the polynucleotide in a virus (or intoa viral vector) and transducing a host cell with the virus (or vector)or by transfection procedures known in the art, as exemplified by U.S.Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which patentsare hereby incorporated herein by reference). The transformationprocedure used depends upon the host to be transformed. Methods forintroducing heterologous polynucleotides into mammalian cells are wellknown in the art and include dextran-mediated transfection, calciumphosphate precipitation, polybrene mediated transfection, protoplastfusion, electroporation, encapsulation of the polynucleotide(s) inliposomes, and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are well known inthe art and include many immortalized cell lines available from theAmerican Type Culture Collection (ATCC), including but not limited toChinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK)cells, monkey kidney cells (COS), human hepatocellular carcinoma cells(e.g., Hep G2), human epithelial kidney 293 cells, and a number of othercell lines. Cell lines of particular preference are selected throughdetermining which cell lines have high expression levels and produceantibodies with constitutive PCSK9 binding properties.

In certain embodiments, antibodies and/or ABP are produced by at leastone of the following hybridomas: 21B12, 31H4, 16F12, any the otherhybridomas listed in Table 2 or disclosed in the examples. In certainembodiments, antigen binding proteins bind to PCSK9 with a dissociationconstant (K_(D)) of less than approximately 1 nM, e.g., 1000 pM to 100pM, 100 pM to 10 pM, 10 pM to 1 pM, and/or 1 pM to 0.1 pM or less.

In certain embodiments, antigen binding proteins comprise animmunoglobulin molecule of at least one of the IgG1, IgG2, IgG3, IgG4,Ig E, IgA, IgD, and IgM isotype. In certain embodiments, antigen bindingproteins comprise a human kappa light chain and/or a human heavy chain.In certain embodiments, the heavy chain is of the IgG1, IgG2, IgG3,IgG4, IgE, IgA, IgD, or IgM isotype. In certain embodiments, antigenbinding proteins have been cloned for expression in mammalian cells. Incertain embodiments, antigen binding proteins comprise a constant regionother than any of the constant regions of the IgG1, IgG2, IgG3, IgG4,IgE, IgA, IgD, and IgM isotype.

In certain embodiments, antigen binding proteins comprise a human lambdalight chain and a human IgG2 heavy chain. In certain embodiments,antigen binding proteins comprise a human lambda light chain and a humanIgG4 heavy chain. In certain embodiments, antigen binding proteinscomprise a human lambda light chain and a human IgG1, IgG3, IgE, IgA,IgD or IgM heavy chain. In other embodiments, antigen binding proteinscomprise a human kappa light chain and a human IgG2 heavy chain. Incertain embodiments, antigen binding proteins comprise a human kappalight chain and a human IgG4 heavy chain. In certain embodiments,antigen binding proteins comprise a human kappa light chain and a humanIgG1, IgG3, IgE, IgA, IgD or IgM heavy chain. In certain embodiments,antigen binding proteins comprise variable regions of antibodies ligatedto a constant region that is neither the constant region for the IgG2isotype, nor the constant region for the IgG4 isotype. In certainembodiments, antigen binding proteins have been cloned for expression inmammalian cells.

In certain embodiments, conservative modifications to the heavy andlight chains of antibodies from at least one of the hybridoma lines:21B12, 31H4 and 16F12 (and corresponding modifications to the encodingnucleotides) will produce antibodies to PCSK9 having functional andchemical characteristics similar to those of the antibodies from thehybridoma lines: 21B12, 31H4 and 16F12. In contrast, in certainembodiments, substantial modifications in the functional and/or chemicalcharacteristics of antibodies to PCSK9 can be accomplished by selectingsubstitutions in the amino acid sequence of the heavy and light chainsthat differ significantly in their effect on maintaining (a) thestructure of the molecular backbone in the area of the substitution, forexample, as a sheet or helical conformation, (b) the charge orhydrophobicity of the molecule at the target site, or (c) the bulk ofthe side chain.

For example, a “conservative amino acid substitution” can involve asubstitution of a native amino acid residue with a normative residuesuch that there is little or no effect on the polarity or charge of theamino acid residue at that position. Furthermore, any native residue inthe polypeptide can also be substituted with alanine, as has beenpreviously described for “alanine scanning mutagenesis.”

Desired amino acid substitutions (whether conservative ornon-conservative) can be determined by those skilled in the art at thetime such substitutions are desired. In certain embodiments, amino acidsubstitutions can be used to identify important residues of antibodiesto PCSK9, or to increase or decrease the affinity of the antibodies toPCSK9 as described herein.

In certain embodiments, antibodies of the present invention can beexpressed in cell lines other than hybridoma cell lines. In certainembodiments, sequences encoding particular antibodies can be used fortransformation of a suitable mammalian host cell. According to certainembodiments, transformation can be by any known method for introducingpolynucleotides into a host cell, including, for example packaging thepolynucleotide in a virus (or into a viral vector) and transducing ahost cell with the virus (or vector) or by transfection procedures knownin the art, as exemplified by U.S. Pat. Nos. 4,399,216, 4,912,040,4,740,461, and 4,959,455 (which patents are hereby incorporated hereinby reference for any purpose). In certain embodiments, thetransformation procedure used can depend upon the host to betransformed. Methods for introduction of heterologous polynucleotidesinto mammalian cells are well known in the art and include, but are notlimited to, dextran-mediated transfection, calcium phosphateprecipitation, polybrene mediated transfection, protoplast fusion,electroporation, encapsulation of the polynucleotide(s) in liposomes,and direct microinjection of the DNA into nuclei.

Mammalian cell lines available as hosts for expression are well known inthe art and include, but are not limited to, many immortalized celllines available from the American Type Culture Collection (ATCC),including but not limited to Chinese hamster ovary (CHO) cells, HeLacells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), humanhepatocellular carcinoma cells (e.g., Hep G2), and a number of othercell lines. In certain embodiments, cell lines can be selected throughdetermining which cell lines have high expression levels and produceantibodies with constitutive HGF binding properties. Appropriateexpression vectors for mammalian host cells are well known.

In certain embodiments, antigen binding proteins comprise one or morepolypeptides. In certain embodiments, any of a variety of expressionvector/host systems can be utilized to express polynucleotide moleculesencoding polypeptides comprising one or more ABP components or the ABPitself. Such systems include, but are not limited to, microorganisms,such as bacteria transformed with recombinant bacteriophage, plasmid, orcosmid DNA expression vectors; yeast transformed with yeast expressionvectors; insect cell systems infected with virus expression vectors(e.g., baculovirus); plant cell systems transfected with virusexpression vectors (e.g., cauliflower mosaic virus, CaMV, tobacco mosaicvirus, TMV) or transformed with bacterial expression vectors (e.g., Tior pBR322 plasmid); or animal cell systems.

In certain embodiments, a polypeptide comprising one or more ABPcomponents or the ABP itself is recombinantly expressed in yeast.Certain such embodiments use commercially available expression systems,e.g., the Pichia Expression System (Invitrogen, San Diego, Calif.),following the manufacturer's instructions. In certain embodiments, sucha system relies on the pre-pro-alpha sequence to direct secretion. Incertain embodiments, transcription of the insert is driven by thealcohol oxidase (AOX1) promoter upon induction by methanol.

In certain embodiments, a secreted polypeptide comprising one or moreABP components or the ABP itself is purified from yeast growth medium.In certain embodiments, the methods used to purify a polypeptide fromyeast growth medium is the same as those used to purify the polypeptidefrom bacterial and mammalian cell supernatants.

In certain embodiments, a nucleic acid encoding a polypeptide comprisingone or more ABP components or the ABP itself is cloned into abaculovirus expression vector, such as pVL1393 (PharMingen, San Diego,Calif.). In certain embodiments, such a vector can be used according tothe manufacturer's directions (PharMingen) to infect Spodopterafrugiperda cells in sF9 protein-free media and to produce recombinantpolypeptide. In certain embodiments, a polypeptide is purified andconcentrated from such media using a heparin-Sepharose column(Pharmacia).

In certain embodiments, a polypeptide comprising one or more ABPcomponents or the ABP itself is expressed in an insect system. Certaininsect systems for polypeptide expression are well known to those ofskill in the art. In one such system, Autographa californica nuclearpolyhedrosis virus (AcNPV) is used as a vector to express foreign genesin Spodoptera frugiperda cells or in Trichoplusia larvae. In certainembodiments, a nucleic acid molecule encoding a polypeptide can beinserted into a nonessential gene of the virus, for example, within thepolyhedrin gene, and placed under control of the promoter for that gene.In certain embodiments, successful insertion of a nucleic acid moleculewill render the nonessential gene inactive. In certain embodiments, thatinactivation results in a detectable characteristic. For example,inactivation of the polyhedrin gene results in the production of viruslacking coat protein.

In certain embodiments, recombinant viruses can be used to infect S.frugiperda cells or Trichoplusia larvae. See, e.g., Smith et al., J.Virol., 46: 584 (1983); Engelhard et al., Proc. Nat. Acad. Sci. (USA),91: 3224-7 (1994).

In certain embodiments, polypeptides comprising one or more ABPcomponents or the ABP itself made in bacterial cells are produced asinsoluble inclusion bodies in the bacteria. In certain embodiments, hostcells comprising such inclusion bodies are collected by centrifugation;washed in 0.15 M NaCl, 10 mM Tris, pH 8, 1 mM EDTA; and treated with 0.1mg/ml lysozyme (Sigma, St. Louis, Mo.) for 15 minutes at roomtemperature. In certain embodiments, the lysate is cleared bysonication, and cell debris is pelleted by centrifugation for 10 minutesat 12,000×g. In certain embodiments, the polypeptide-containing pelletis resuspended in 50 mM Tris, pH 8, and 10 mM EDTA; layered over 50%glycerol; and centrifuged for 30 minutes at 6000×g. In certainembodiments, that pellet can be resuspended in standard phosphatebuffered saline solution (PBS) free of Mg⁺⁺ and Ca⁺⁺. In certainembodiments, the polypeptide is further purified by fractionating theresuspended pellet in a denaturing SDS polyacrylamide gel (See, e.g.,Sambrook et al., supra). In certain embodiments, such a gel can besoaked in 0.4 M KCl to visualize the protein, which can be excised andelectroeluted in gel-running buffer lacking SDS. According to certainembodiments, a Glutathione-S-Transferase (GST) fusion protein isproduced in bacteria as a soluble protein. In certain embodiments, suchGST fusion protein is purified using a GST Purification Module(Pharmacia).

In certain embodiments, it is desirable to “refold” certainpolypeptides, e.g., polypeptides comprising one or more ABP componentsor the ABP itself. In certain embodiments, such polypeptides areproduced using certain recombinant systems discussed herein. In certainembodiments, polypeptides are “refolded” and/or oxidized to form desiredtertiary structure and/or to generate disulfide linkages. In certainembodiments, such structure and/or linkages are related to certainbiological activity of a polypeptide. In certain embodiments, refoldingis accomplished using any of a number of procedures known in the art.Exemplary methods include, but are not limited to, exposing thesolubilized polypeptide agent to a pH typically above 7 in the presenceof a chaotropic agent. An exemplary chaotropic agent is guanidine. Incertain embodiments, the refolding/oxidation solution also contains areducing agent and the oxidized form of that reducing agent. In certainembodiments, the reducing agent and its oxidized form are present in aratio that will generate a particular redox potential that allowsdisulfide shuffling to occur. In certain embodiments, such shufflingallows the formation of cysteine bridges. Exemplary redox couplesinclude, but are not limited to, cysteine/cystamine,glutathione/dithiobisGSH, cupric chloride, dithiothreitol DTT/dithianeDTT, and 2-mercaptoethanol (bME)/dithio-bME. In certain embodiments, aco-solvent is used to increase the efficiency of refolding. Exemplarycosolvents include, but are not limited to, glycerol, polyethyleneglycol of various molecular weights, and arginine.

In certain embodiments, one substantially purifies a polypeptidecomprising one or more ABP components or the ABP itself. Certain proteinpurification techniques are known to those of skill in the art. Incertain embodiments, protein purification involves crude fractionationof polypeptide fractionations from non-polypeptide fractions. In certainembodiments, polypeptides are purified using chromatographic and/orelectrophoretic techniques. Exemplary purification methods include, butare not limited to, precipitation with ammonium sulphate; precipitationwith PEG; immunoprecipitation; heat denaturation followed bycentrifugation; chromatography, including, but not limited to, affinitychromatography (e.g., Protein-A-Sepharose), ion exchange chromatography,exclusion chromatography, and reverse phase chromatography; gelfiltration; hydroxyapatite chromatography; isoelectric focusing;polyacrylamide gel electrophoresis; and combinations of such and othertechniques. In certain embodiments, a polypeptide is purified by fastprotein liquid chromatography or by high pressure liquid chromotography(HPLC). In certain embodiments, purification steps can be changed orcertain steps can be omitted, and still result in a suitable method forthe preparation of a substantially purified polypeptide.

In certain embodiments, one quantitates the degree of purification of apolypeptide preparation. Certain methods for quantifying the degree ofpurification are known to those of skill in the art. Certain exemplarymethods include, but are not limited to, determining the specificbinding activity of the preparation and assessing the amount of apolypeptide within a preparation by SDS/PAGE analysis. Certain exemplarymethods for assessing the amount of purification of a polypeptidepreparation comprise calculating the binding activity of a preparationand comparing it to the binding activity of an initial extract. Incertain embodiments, the results of such a calculation are expressed as“fold purification.” The units used to represent the amount of bindingactivity depend upon the particular assay performed.

In certain embodiments, a polypeptide comprising one or more ABPcomponents or the ABP itself is partially purified. In certainembodiments, partial purification can be accomplished by using fewerpurification steps or by utilizing different forms of the same generalpurification scheme. For example, in certain embodiments,cation-exchange column chromatography performed utilizing an HPLCapparatus will generally result in a greater “fold purification” thanthe same technique utilizing a low-pressure chromatography system. Incertain embodiments, methods resulting in a lower degree of purificationcan have advantages in total recovery of polypeptide, or in maintainingbinding activity of a polypeptide.

In certain instances, the electrophoretic migration of a polypeptide canvary, sometimes significantly, with different conditions of SDS/PAGE.See, e.g., Capaldi et al., Biochem. Biophys. Res. Comm., 76: 425 (1977).It will be appreciated that under different electrophoresis conditions,the apparent molecular weights of purified or partially purifiedpolypeptide can be different.

Exemplary Epitopes

Epitopes to which anti-PCSK9 antibodies bind are provided. In someembodiments, epitopes that are bound by the presently disclosedantibodies are particularly useful. In some embodiments, antigen bindingproteins that bind to any of the epitopes that are bound by theantibodies described herein are useful. In some embodiments, theepitopes bound by any of the antibodies listed in Table 2 and FIGS. 2and 3 are especially useful. In some embodiments, the epitope is on thecatalytic domain PCSK9.

In certain embodiments, a PCSK9 epitope can be utilized to prevent(e.g., reduce) binding of an anti-PCSK9 antibody or antigen bindingprotein to PCSK9. In certain embodiments, a PCSK9 epitope can beutilized to decrease binding of an anti-PCSK9 antibody or antigenbinding protein to PCSK9. In certain embodiments, a PCSK9 epitope can beutilized to substantially inhibit binding of an anti-PCSK9 antibody orantigen binding protein to PCSK9.

In certain embodiments, a PCSK9 epitope can be utilized to isolateantibodies or antigen binding proteins that bind to PCSK9. In certainembodiments, a PCSK9 epitope can be utilized to generate antibodies orantigen binding proteins which bind to PCSK9. In certain embodiments, aPCSK9 epitope or a sequence comprising a PCSK9 epitope can be utilizedas an immunogen to generate antibodies or antigen binding proteins thatbind to PCSK9. In certain embodiments, a PCSK9 epitope can beadministered to an animal, and antibodies that bind to PCSK9 cansubsequently be obtained from the animal. In certain embodiments, aPCSK9 epitope or a sequence comprising a PCSK9 epitope can be utilizedto interfere with normal PCSK9-mediated activity, such as association ofPCSK9 with the LDLR.

In some embodiments, antigen binding proteins disclosed herein bindspecifically to N-terminal prodomain, a subtilisin-like catalytic domainand/or a C-terminal domain. In some embodiments, the antigen bindingprotein binds to the substrate-binding groove of PCSK-9 (described inCunningham et al., incorporated herein in its entirety by reference).

In some embodiments, the domain(s)/region(s) containing residues thatare in contact with or are buried by an antibody can be identified bymutating specific residues in PCSK9 (e.g., a wild-type antigen) anddetermining whether the antigen binding protein can bind the mutated orvariant PCSK9 protein. By making a number of individual mutations,residues that play a direct role in binding or that are in sufficientlyclose proximity to the antibody such that a mutation can affect bindingbetween the antigen binding protein and antigen can be identified. Froma knowledge of these amino acids, the domain(s) or region(s) of theantigen that contain residues in contact with the antigen bindingprotein or covered by the antibody can be elucidated. Such a domain caninclude the binding epitope of an antigen binding protein. One specificexample of this general approach utilizes an arginine/glutamic acidscanning protocol (see, e.g., Nanevicz, T., et al., 1995, J. Biol.Chem., 270:37, 21619-21625 and Zupnick, A., et al., 2006, J. Biol.Chem., 281:29, 20464-20473). In general, arginine and glutamic acids aresubstituted (typically individually) for an amino acid in the wild-typepolypeptide because these amino acids are charged and bulky and thushave the potential to disrupt binding between an antigen binding proteinand an antigen in the region of the antigen where the mutation isintroduced. Arginines that exist in the wild-type antigen are replacedwith glutamic acid. A variety of such individual mutants are obtainedand the collected binding results analyzed to determine what residuesaffect binding.

Example 39 describes one such arginine/glutamic acid scanning of PCSK9for PCSK9 antigen binding proteins provided herein. A series of mutantPCSK9 antigens were created, with each mutant antigen having a singlemutation. Binding of each mutant PCSK9 antigen with various PCSK9 ABPswas measured and compared to the ability of the selected ABPs to bindwild-type PCSK9 (SEQ ID NO: 303).

An alteration (for example a reduction or increase) in binding betweenan antigen binding protein and a variant PCSK9 as used herein means thatthere is a change in binding affinity (e.g., as measured by knownmethods such as Biacore testing or the bead based assay described belowin the examples), EC₅₀, and/or a change (for example a reduction) in thetotal binding capacity of the antigen binding protein (for example, asevidenced by a decrease in Bmax in a plot of antigen binding proteinconcentration versus antigen concentration). A significant alteration inbinding indicates that the mutated residue is directly involved inbinding to the antigen binding protein or is in close proximity to thebinding protein when the binding protein is bound to antigen.

In some embodiments, a significant reduction in binding means that thebinding affinity, EC50, and/or capacity between an antigen bindingprotein and a mutant PCSK9 antigen is reduced by greater than 10%,greater than 20%, greater than 40%, greater than 50%, greater than 55%,greater than 60%, greater than 65%, greater than 70%, greater than 75%,greater than 80%, greater than 85%, greater than 90% or greater than 95%relative to binding between the antigen binding protein and a wild typePCSK9 (e.g., shown in SEQ ID NO: 1 and/or SEQ ID NO: (303). In certainembodiments, binding is reduced below detectable limits. In someembodiments, a significant reduction in binding is evidenced whenbinding of an antigen binding protein to a variant PCSK9 protein is lessthan 50% (for example, less than 40%, 35%, 30%, 25%, 20%, 15% or 10%) ofthe binding observed between the antigen binding protein and a wild-typePCSK9 protein (for example, the protein of SEQ ID NO: 1 and/or SEQ IDNO: (303). Such binding measurements can be made using a variety ofbinding assays known in the art. A specific example of one such assay isdescribed in Example 39.

In some embodiments, antigen binding proteins are provided that exhibitsignificantly lower binding for a variant PCSK9 protein in which aresidue in a wild-type PCSK9 protein (e.g., SEQ ID NO: 1 or SEQ ID NO:303 is substituted with arginine or glutamic acid. In some embodiments,binding of an antigen binding protein is significantly reduced orincreased for a variant PCSK9 protein having any one or more (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or 244) of the following mutations: R207E,D208R, R185E, R439E, E513R, V538R, E539R, T132R, S351R, A390R, A413R,E582R, D162R, R164E, E167R, S123R, E129R, A311R, D313R, D337R, R519E,H521R, and Q554R as compared to a wild-type PCSK9 protein (e.g., SEQ IDNO: 1 or SEQ ID NO: 303. In the shorthand notation used here, the formatis: Wild type residue: Position in polypeptide: Mutant residue, with thenumbering of the residues as indicated in SEQ ID NO: 1 or SEQ ID NO:303.

In some embodiments, binding of an antigen binding protein issignificantly reduced or increased for a mutant PCSK9 protein having oneor more (e.g., 1, 2, 3, 4, 5, or more) mutations at the followingpositions: 207, 208, 185, 181, 439, 513, 538, 539, 132, 351, 390, 413,582, 162, 164, 167, 123, 129, 311, 313, 337, 519, 521, and 554, as shownin SEQ ID NO: 1 as compared to a wild-type PCSK9 protein (e.g., SEQ IDNO: 1 or SEQ ID NO: 303. In some embodiments, binding of an antigenbinding protein is reduced or increased for a mutant PCSK9 proteinhaving one or more (e.g., 1, 2, 3, 4, 5, or more) mutations at thefollowing positions: 207, 208, 185, 181, 439, 513, 538, 539, 132, 351,390, 413, 582, 162, 164, 167, 123, 129, 311, 313, 337, 519, 521, and554, as shown in SEQ ID NO: 1 as compared to a wild-type PCSK9 protein(e.g., SEQ ID NO: 1 or SEQ ID NO: 303. In some embodiments, binding ofan antigen binding protein is substantially reduced or increased for amutant PCSK9 protein having one or more (e.g., 1, 2, 3, 4, 5, or more)mutations at the following positions: 207, 208, 185, 181, 439, 513, 538,539, 132, 351, 390, 413, 582, 162, 164, 167, 123, 129, 311, 313, 337,519, 521, and 554, within SEQ ID NO: 1 as compared to a wild-type PCSK9protein (e.g., SEQ ID NO: 1 or SEQ ID NO: 303.

In some embodiments, binding of an ABP is significantly reduced orincreased for a mutant PCSK9 protein having one or more (e.g., 1, 2, 3,4, 5, etc.) of the following mutations: R207E, D208R, R185E, R439E,E513R, V538R, E539R, T132R, S351R, A390R, A413R, E582R, D162R, R164E,E167R, S123R, E129R, A311R, D313R, D337R, R519E, H521R, and Q554R withinSEQ ID NO: 1 or SEQ ID NO: 303, as compared to a wild-type PCSK9 protein(e.g., SEQ ID NO: 1 or SEQ ID NO: 303).

In some embodiments, binding of an ABP is significantly reduced orincreased for a mutant PCSK9 protein having one or more (e.g., 1, 2, 3,4, 5, etc.) of the following mutations: R207E, D208R, R185E, R439E,E513R, V538R, E539R, T132R, S351R, A390R, A413R, and E582R within SEQ IDNO: 1 or SEQ ID NO: 303, as compared to a wild-type PCSK9 protein (e.g.,SEQ ID NO: 1 or SEQ ID NO: 303). In some embodiments, the binding isreduced. In some embodiments, the reduction in binding is observed as achange in EC50. In some embodiments, the change in EC50 is an increasein the numerical value of the EC50 (and thus is a decrease in binding).

In some embodiments, binding of an ABP is significantly reduced orincreased for a mutant PCSK9 protein having one or more (e.g., 1, 2, 3,4, 5, etc.) of the following mutations: D162R, R164E, E167R, S123R,E129R, A311R, D313R, D337R, R519E, H521R, and Q554R within SEQ ID NO: 1,as compared to a wild-type PCSK9 protein (e.g., SEQ ID NO: 1 or SEQ IDNO: 303). In some embodiments, the binding is reduced. In someembodiments, the reduction in binding is observed as a change in Bmax.In some embodiments, the shift in Bmax is a reduction of the maximumsignal generated by the ABP. In some embodiments, for an amino acid tobe part of an epitope, the Bmax is reduced by at least 10%, for example,reductions of at least any of the following amounts: 20, 30, 40, 50, 60,70, 80, 90, 95, 98, 99, or 100 percent can, in some embodiments,indicate that the residue is part of the epitope.

Although the variant forms just listed are referenced with respect tothe wild-type sequence shown in SEQ ID NO: 1 or SEQ ID NO: 303, it willbe appreciated that in an allelic variant of PCSK9 the amino acid at theindicated position could differ. Antigen binding proteins showingsignificantly lower binding for such allelic forms of PCSK9 are alsocontemplated. Accordingly, in some embodiments, any of the aboveembodiments can be compared to an allelic sequence, rather than purelythe wild-type sequence shown in FIG. 1A

In some embodiments, binding of an antigen binding protein issignificantly reduced for a variant PCSK9 protein in which the residueat a selected position in the wild-type PCSK9 protein is mutated to anyother residue. In some embodiments, the herein describedarginine/glutamic acid replacements are used for the identifiedpositions. In some embodiments, alanine is used for the identifiedpositions.

As noted above, residues directly involved in binding or covered by anantigen binding protein can be identified from scanning results. Theseresidues can thus provide an indication of the domains or regions of SEQID NO: 1 (or SEQ ID NO: 303 or SEQ ID NO: 3) that contain the bindingregion(s) to which antigen binding proteins bind. As can be seen fromthe results summarized in Example 39, in some embodiments an antigenbinding protein binds to a domain containing at least one of aminoacids: 207, 208, 185, 181, 439, 513, 538, 539, 132, 351, 390, 413, 582,162, 164, 167, 123, 129, 311, 313, 337, 519, 521, and 554 of SEQ ID NO:1 or SEQ ID NO: 303. In some embodiments, the antigen binding proteinbinds to a region containing at least one of amino acids 207, 208, 185,181, 439, 513, 538, 539, 132, 351, 390, 413, 582, 162, 164, 167, 123,129, 311, 313, 337, 519, 521, and 554 of SEQ ID NO: 1 or SEQ ID NO: 303.

In some embodiments, the antigen binding protein binds to a regioncontaining at least one of amino acids 162, 164, 167, 207 and/or 208 ofSEQ ID NO: 1 or SEQ ID NO: 303. In some embodiments, more than one(e.g., 2, 3, 4, or 5) of the identified residues are part of the regionthat is bound by the ABP. In some embodiments, the ABP competes with ABP21B12.

In some embodiments, the antigen binding protein binds to a regioncontaining at least one of amino acid 185 of SEQ ID NO: 1 or SEQ ID NO:303. In some embodiments, the ABP competes with ABP 31H4.

In some embodiments, the antigen binding protein binds to a regioncontaining at least one of amino acids 439, 513, 538, and/or 539 of SEQID NO: 1 or SEQ ID NO: 303. In some embodiments, more than one (e.g., 2,3, or 4) of the identified residues are part of the region that is boundby the ABP. In some embodiments, the ABP competes with ABP 31A4.

In some embodiments, the antigen binding protein binds to a regioncontaining at least one of amino acids 123, 129, 311, 313, 337, 132,351, 390, and/or 413 of SEQ ID NO: 1 or SEQ ID NO: 303. In someembodiments, more than one (e.g., 2, 3, 4, 5, 6, 7, 8, or 9) of theidentified residues are part of the region that is bound by the ABP. Insome embodiments, the ABP competes with ABP 12H11.

In some embodiments, the antigen binding protein binds to a regioncontaining at least one of amino acid 582, 519, 521, and/or 554 of SEQID NO: 1 or SEQ ID NO: 303. In some embodiments, more than one (e.g., 2,3, or 4) of the identified residues are part of the region that is boundby the ABP. In some embodiments, the ABP competes with ABP 3C4.

In some embodiments, the antigen binding proteins binds to the foregoingregions within a fragment or the full length sequence of SEQ ID NO: 1 orSEQ ID NO: 303. In other embodiments, antigen binding proteins bind topolypeptides consisting of these regions. The reference to “SEQ ID NO: 1or SEQ ID NO: 303” denotes that one or both of these sequences can beemployed or relevant. The phrase does not denote that only one should beemployed.

As noted above, the above description references specific amino acidpositions with reference to SEQ ID NO: 1. However, throughout thespecification generally, reference is made to a Pro/Cat domain thatcommences at position 31, which is provided in SEQ ID NO: 3. As notedbelow, SEQ ID NO: 1 and SEQ ID NO: 303 lack the signal sequence ofPCSK9. As such, any comparison between these various disclosures shouldtake this difference in numbering into account. In particular, any aminoacid position in SEQ ID NO: 1, will correspond to an amino acid position30 amino acids further into the protein in SEQ ID NO: 3. For example,position 207 of SEQ ID NO: 1, corresponds to position 237 of SEQ ID NO:3 (the full length sequence, and the numbering system used in thepresent specification generally). Table 39.6 outlines how the abovenoted positions, which reference SEQ ID NO: 1 (and/or SEQ ID NO: 303)correspond to SEQ ID NO: 3 (which includes the signal sequence). Thus,any of the above noted embodiments that are described in regard to SEQID NO: 1 (and/or SEQ ID NO: 303), are described in reference to SEQ IDNO: 3, by the noted corresponding positions.

In some embodiments, ABP 21B12 binds to an epitope including residues162-167 (e.g., residues D162-E167 of SEQ ID NO: 1). In some embodiments,ABP 12H11 binds to an epitope that includes residues 123-132 (e.g.,S123-T132 of SEQ ID NO: 1). In some embodiments, ABP 12H11 binds to anepitope that includes residues 311-313 (e.g., A311-D313 of SEQ ID NO:1). In some embodiments, ABPs can bind to an epitope that includes anyone of these strands of sequences.

Competing Antigen Binding Proteins

In another aspect, antigen binding proteins are provided that competewith one of the exemplified antibodies or functional fragments bindingto the epitope described herein for specific binding to PCSK9. Suchantigen binding proteins can also bind to the same epitope as one of theherein exemplified antigen binding proteins, or an overlapping epitope.Antigen binding proteins and fragments that compete with or bind to thesame epitope as the exemplified antigen binding proteins are expected toshow similar functional properties. The exemplified antigen bindingproteins and fragments include those described above, including thosewith the heavy and light chains, variable region domains and CDRsincluded in TABLE 2 And/or FIGS. 2-3 and 15. Thus, as a specificexample, the antigen binding proteins that are provided include thosethat compete with an antibody or antigen binding protein having:

(a) all 6 of the CDRs listed for an antibody listed in FIGS. 2-3 and 15;

(b) a VH and a VL listed for an antibody listed in Table 2; or

(c) two light chains and two heavy chains as specified for an antibodylisted in Table 2.

Certain Therapeutic Uses and Pharmaceutical Compositions

In certain instances, PCSK9 activity correlates with a number of humandisease states. For example, in certain instances, too much or toolittle PCSK9 activity correlates with certain conditions, such ashypercholesterolemia. Therefore, in certain instances, modulating PCSK9activity can be therapeutically useful. In certain embodiments, aneutralizing antigen binding protein to PCSK9 is used to modulate atleast one PCSK9 activity (e.g., binding to LDLR). Such methods can treatand/or prevent and/or reduce the risk of disorders that relate toelevated serum cholesterol levels or in which elevated cholesterollevels are relevant.

As will be appreciated by one of skill in the art, in light of thepresent disclosure, disorders that relate to, involve, or can beinfluenced by varied cholesterol, LDL, or LDLR levels can be addressedby various embodiments of the antigen binding proteins. In someembodiments, a “cholesterol related disorder” (which includes “serumcholesterol related disorders”) includes any one or more of thefollowing: hypercholesterolemia, heart disease, metabolic syndrome,diabetes, coronary heart disease, stroke, cardiovascular diseases,Alzheimers disease and generally dyslipidemias, which can be manifested,for example, by an elevated total serum cholesterol, elevated LDL,elevated triglycerides, elevated VLDL, and/or low HDL. Some non-limitingexamples of primary and secondary dyslipidemias that can be treatedusing an ABP, either alone, or in combination with one or more otheragents include the metabolic syndrome, diabetes mellitus, familialcombined hyperlipidemia, familial hypertriglyceridemia, familialhypercholesterolemias, including heterozygous hypercholesterolemia,homozygous hypercholesterolemia, familial defective apoplipoproteinB-100; polygenic hypercholesterolemia; remnant removal disease, hepaticlipase deficiency; dyslipidemia secondary to any of the following:dietary indiscretion, hypothyroidism, drugs including estrogen andprogestin therapy, beta-blockers, and thiazide diuretics; nephroticsyndrome, chronic renal failure, Cushing's syndrome, primary biliarycirrhosis, glycogen storage diseases, hepatoma, cholestasis, acromegaly,insulinoma, isolated growth hormone deficiency, and alcohol-inducedhypertriglyceridemia. ABP can also be useful in preventing or treatingatherosclerotic diseases, such as, for example, coronary heart disease,coronary artery disease, peripheral arterial disease, stroke (ischaemicand hemorrhagic), angina pectoris, or cerebrovascular disease and acutecoronary syndrome, myocardial infarction. In some embodiments, the ABPis useful in reducing the risk of: nonfatal heart attacks, fatal andnon-fatal strokes, certain types of heart surgery, hospitalization forheart failure, chest pain in patients with heart disease, and/orcardiovascular events because of established heart disease such as priorheart attack, prior heart surgery, and/or chest pain with evidence ofclogged arteries. In some embodiments, the ABP and methods can be usedto reduce the risk of recurrent cardiovascular events.

As will be appreciated by one of skill in the art, diseases or disordersthat are generally addressable (either treatable or preventable) throughthe use of statins can also benefit from the application of the instantantigen binding proteins. In addition, in some embodiments, disorders ordisease that can benefit from the prevention of cholesterol synthesis orincreased LDLR expression can also be treated by various embodiments ofthe antigen binding proteins. In addition, as will be appreciated by oneof skill in the art, the use of the anti-PCSK9 antibodies can beespecially useful in the treatment of Diabetes. Not only is Diabetes arisk factor for coronary heart disease, but insulin increases theexpression of PCSK9. That is, people with Diabetes have elevated plasmalipid levels (which can be related to high PCSK9 levels) and can benefitfrom lowering those levels. This is generally discussed in more detailin Costet et al. (“Hepatic PCSK9 Expression is Regulated by NutirtionalStatus via Insulin and Sterol Regulatiory Element-binding Protein 1C”,J. Biol. Chem., 281: 6211-6218, 2006), the entirety of which isincorporated herein by reference.

In some embodiments, the antigen binding protein is administered tothose who have diabetes mellitus, abdominal aortic aneurysm,atherosclerosis and/or peripheral vascular disease in order to decreasetheir serum cholesterol levels to a safer range. In some embodiments,the antigen binding protein is administered to patients at risk ofdeveloping any of the herein described disorders. In some embodiments,the ABPs are administered to subjects that smoke, have hypertension or afamilial history of early heart attacks.

In some embodiments, a subject is administered an ABP if they are at amoderate risk or higher on the 2004 NCEP treatment goals. In someembodiments, the ABP is administered to a subject if the subject's LDLcholesterol level is greater than 160 mg/dl. In some embodiments, theABP is administered if the subjects LDL cholesterol level is greaterthan 130 (and they have a moderate or moderately high risk according tothe 2004 NCEP treatment goals). In some embodiments, the ABP isadministered if the subjects LDL cholesterol level is greater than 100(and they have a high or very high risk according to the 2004 NCEPtreatment goals).

A physician will be able to select an appropriate treatment indicationsand target lipid levels depending on the individual profile of aparticular patient. One well-accepted standard for guiding treatment ofhyperlipidemia is the Third Report of the National Cholesterol EducationProgram (NCEP) Expert Panel on Detection, Evaluation, and Treatment ofthe High Blood Cholesterol in Adults (Adult Treatment Panel III) FinalReport, National Institutes of Health, NIH Publication No. 02-5215(2002), the printed publication of which is hereby incorporated byreference in its entirety.

In some embodiments, antigen binding proteins to PCSK9 are used todecrease the amount of PCSK9 activity from an abnormally high level oreven a normal level. In some embodiments, antigen binding proteins toPCSK9 are used to treat or prevent hypercholesterolemia and/or in thepreparation of medicaments therefore and/or for other cholesterolrelated disorders (such as those noted herein). In certain embodiments,an antigen binding protein to PCSK9 is used to treat or preventconditions such as hypercholesterolemia in which PCSK9 activity isnormal. In such conditions, for example, reduction of PCSK9 activity tobelow normal can provide a therapeutic effect.

In some embodiments, more than one antigen binding protein to PCSK9 isused to modulate PCSK9 activity.

In certain embodiments, methods are provided of treating a cholesterolrelated disorder, such as hypercholesterolemia comprising administeringa therapeutically effective amount of one or more antigen bindingproteins to PCSK9 and another therapeutic agent.

In certain embodiments, an antigen binding protein to PCSK9 isadministered alone. In certain embodiments, an antigen binding proteinto PCSK9 is administered prior to the administration of at least oneother therapeutic agent. In certain embodiments, an antigen bindingprotein to PCSK9 is administered concurrent with the administration ofat least one other therapeutic agent. In certain embodiments, an antigenbinding protein to PCSK9 is administered subsequent to theadministration of at least one other therapeutic agent. In otherembodiments, an antigen binding protein to PCSK9 is administered priorto the administration of at least one other therapeutic agent.Therapeutic agents (apart from the antigen binding protein), include,but are not limited to, at least one other cholesterol-lowering (serumand/or total body cholesterol) agent or an agent. In some embodiments,the agent increases the expression of LDLR, have been observed toincrease serum HDL levels, lower LDL levels or lower triglyceridelevels. Exemplary agents include, but are not limited to, statins(atorvastatin, cerivastatin, fluvastatin, lovastatin, mevastatin,pitavastatin, pravastatin, rosuvastatin, simvastatin), Nicotinic acid(Niacin) (NIACOR, NIASPAN (slow release niacin), SLO-NIACIN (slowrelease niacin)), Fibric acid (LOPID (Gemfibrozil), TRICOR(fenofibrate), Bile acid sequestrants (QUESTRAN (cholestyramine),colesevelam (WELCHOL), COLESTID (colestipol)), Cholesterol absorptioninhibitors (ZETIA (ezetimibe)), Combining nicotinic acid with statin(ADVICOR (LOVASTATIN and NIASPAN), Combining a statin with an absorptioninhibitor (VYTORIN (ZOCOR and ZETIA) and/or lipid modifying agents. Insome embodiments, the ABP is combined with PPAR gamma agonsits, PPARalpha/gamma agonists, squalene synthase inhibitors, CETP inhibitors,anti-hypertensives, anti-diabetic agents (such as sulphonyl ureas,insulin, GLP-1 analogs, DDPIV inhibitors), ApoB modulators, MTPinhibitoris and/or arteriosclerosis obliterans treatments. In someembodiments, the ABP is combined with an agent that increases the levelof LDLR protein in a subject, such as statins, certain cytokines likeoncostatin M, estrogen, and/or certain herbal ingredients such asberberine. In some embodiments, the ABP is combined with an agent thatincreases serum cholesterol levels in a subject (such as certainanti-psycotic agents, certain HIV protease inhibitors, dietary factorssuch as high fructose, sucrose, cholesterol or certain fatty acids andcertain nuclear receptor agonists and antagonists for RXR, RAR, LXR,FXR). In some embodiments, the ABP is combined with an agent thatincreases the level of PCSK9 in a subject, such as statins and/orinsulin. The combination of the two can allow for the undesirableside-effects of other agents to be mitigated by the ABP. As will beappreciated by one of skill in the art, in some embodiments, the ABP iscombined with the other agent/compound. In some embodiments, the ABP andother agent are administered concurrently. In some embodiments, the ABPand other agent are not administered simultaneously, with the ABP beingadministered before or after the agent is administered. In someembodiments, the subject receives both the ABP and the other agent (thatincreases the level of LDLR) during a same period of prevention,occurrence of a disorder, and/or period of treatment.

Pharmaceutical compositions of the invention can be administered incombination therapy, i.e., combined with other agents. In certainembodiments, the combination therapy comprises an antigen bindingprotein capable of binding PCSK9, in combination with at least oneanti-cholesterol agent. Agents include, but are not limited to, in vitrosynthetically prepared chemical compositions, antibodies, antigenbinding regions, and combinations and conjugates thereof. In certainembodiments, an agent can act as an agonist, antagonist, alllostericmodulator, or toxin. In certain embodiments, an agent can act to inhibitor stimulate its target (e.g., receptor or enzyme activation orinhibition), and thereby promote increased expression of LDLR ordecrease serum cholesterol levels.

In certain embodiments, an antigen binding protein to PCSK9 can beadministered prior to, concurrent with, and subsequent to treatment witha cholesterol-lowering (serum and/or total cholesterol) agent. Incertain embodiments, an antigen binding protein to PCSK9 can beadministered prophylactically to prevent or mitigate the onset ofhypercholesterolemia, heart disease, diabetes, and/or any of thecholesterol related disorder. In certain embodiments, an antigen bindingprotein to PCSK9 can be administered for the treatment of an existinghypercholesterolemia condition. In some embodiments, the ABP delays theonset of the disorder and/or symptoms associated with the disorder. Insome embodiments, the ABP is provided to a subject lacking any symptomsof any one of the cholesterol related disorders or a subset thereof.

In certain embodiments, an antigen binding protein to PCSK9 is used withparticular therapeutic agents to treat various cholesterol relateddisorders, such as hypercholesterolemia. In certain embodiments, in viewof the condition and the desired level of treatment, two, three, or moreagents can be administered. In certain embodiments, such agents can beprovided together by inclusion in the same formulation. In certainembodiments, such agent(s) and an antigen binding protein to PCSK9 canbe provided together by inclusion in the same formulation. In certainembodiments, such agents can be formulated separately and providedtogether by inclusion in a treatment kit. In certain embodiments, suchagents and an antigen binding protein to PCSK9 can be formulatedseparately and provided together by inclusion in a treatment kit. Incertain embodiments, such agents can be provided separately. In certainembodiments, when administered by gene therapy, the genes encodingprotein agents and/or an antigen binding protein to PCSK9 can beincluded in the same vector. In certain embodiments, the genes encodingprotein agents and/or an antigen binding protein to PCSK9 can be underthe control of the same promoter region. In certain embodiments, thegenes encoding protein agents and/or an antigen binding protein to PCSK9can be in separate vectors.

In certain embodiments, the invention provides for pharmaceuticalcompositions comprising an antigen binding protein to PCSK9 togetherwith a pharmaceutically acceptable diluent, carrier, solubilizer,emulsifier, preservative and/or adjuvant.

In certain embodiments, the invention provides for pharmaceuticalcompositions comprising an antigen binding protein to PCSK9 and atherapeutically effective amount of at least one additional therapeuticagent, together with a pharmaceutically acceptable diluent, carrier,solubilizer, emulsifier, preservative and/or adjuvant.

In certain embodiments, an antigen binding protein to PCSK9 can be usedwith at least one therapeutic agent for inflammation. In certainembodiments, an antigen binding protein to PCSK9 can be used with atleast one therapeutic agent for an immune disorder. Exemplarytherapeutic agents for inflammation and immune disorders include, butare not limited to cyclooxygenase type 1 (COX-1) and cyclooxygenase type2 (COX-2) inhibitors small molecule modulators of 38 kDamitogen-activated protein kinase (p38-MAPK); small molecule modulatorsof intracellular molecules involved in inflammation pathways, whereinsuch intracellular molecules include, but are not limited to, jnk, IKK,NF-κB, ZAP70, and lck. Certain exemplary therapeutic agents forinflammation are described, e.g., in C. A. Dinarello & L. L. MoldawerProinflammatory and Anti-Inflammatory Cytokines in Rheumatoid Arthritis:A Primer for Clinicians Third Edition (2001) Amgen Inc. Thousand Oaks,Calif.

In certain embodiments, pharmaceutical compositions will include morethan one different antigen binding protein to PCSK9. In certainembodiments, pharmaceutical compositions will include more than oneantigen binding protein to PCSK9 wherein the antigen binding proteins toPCSK9 bind more than one epitope. In some embodiments, the variousantigen binding proteins will not compete with one another for bindingto PCSK9. In some embodiments, any of the antigen binding proteinsdepicted in Table 2 and FIGS. 2 and/or 3 can be combined together in apharmaceutical composition.

In certain embodiments, acceptable formulation materials preferably arenontoxic to recipients at the dosages and concentrations employed. Insome embodiments, the formulation material(s) are for s.c. and/or I.V.administration. In certain embodiments, the pharmaceutical compositioncan contain formulation materials for modifying, maintaining orpreserving, for example, the pH, osmolarity, viscosity, clarity, color,isotonicity, odor, sterility, stability, rate of dissolution or release,adsorption or penetration of the composition. In certain embodiments,suitable formulation materials include, but are not limited to, aminoacids (such as glycine, glutamine, asparagine, arginine or lysine);antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite orsodium hydrogen-sulfite); buffers (such as borate, bicarbonate,Tris-HCl, citrates, phosphates or other organic acids); bulking agents(such as mannitol or glycine); chelating agents (such as ethylenediaminetetraacetic acid (EDTA)); complexing agents (such as caffeine,polyvinylpyrrolidone, beta-cyclodextrin orhydroxypropyl-beta-cyclodextrin); fillers; monosaccharides;disaccharides; and other carbohydrates (such as glucose, mannose ordextrins); proteins (such as serum albumin, gelatin or immunoglobulins);coloring, flavoring and diluting agents; emulsifying agents; hydrophilicpolymers (such as polyvinylpyrrolidone); low molecular weightpolypeptides; salt-forming counterions (such as sodium); preservatives(such as benzalkonium chloride, benzoic acid, salicylic acid,thimerosal, phenethyl alcohol, methylparaben, propylparaben,chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such asglycerin, propylene glycol or polyethylene glycol); sugar alcohols (suchas mannitol or sorbitol); suspending agents; surfactants or wettingagents (such as pluronics, PEG, sorbitan esters, polysorbates such aspolysorbate 20, polysorbate 80, triton, tromethamine, lecithin,cholesterol, tyloxapal); stability enhancing agents (such as sucrose orsorbitol); tonicity enhancing agents (such as alkali metal halides,preferably sodium or potassium chloride, mannitol sorbitol); deliveryvehicles; diluents; excipients and/or pharmaceutical adjuvants.(Remington's Pharmaceutical Sciences, 18^(th) Edition, A. R. Gennaro,ed., Mack Publishing Company (1995). In some embodiments, theformulation comprises PBS; 20 mM NaOAC, pH 5.2, 50 mM NaCl; and/or 10 mMNAOAC, pH 5.2, 9% Sucrose.

In certain embodiments, an antigen binding protein to PCSK9 and/or atherapeutic molecule is linked to a half-life extending vehicle known inthe art. Such vehicles include, but are not limited to, polyethyleneglycol, glycogen (e.g., glycosylation of the ABP), and dextran. Suchvehicles are described, e.g., in U.S. application Ser. No. 09/428,082,now U.S. Pat. No. 6,660,843 and published PCT Application No. WO99/25044, which are hereby incorporated by reference for any purpose.

In certain embodiments, the optimal pharmaceutical composition will bedetermined by one skilled in the art depending upon, for example, theintended route of administration, delivery format and desired dosage.See, for example, Remington's Pharmaceutical Sciences, supra. In certainembodiments, such compositions may influence the physical state,stability, rate of in vivo release and rate of in vivo clearance of theantibodies of the invention.

In certain embodiments, the primary vehicle or carrier in apharmaceutical composition can be either aqueous or non-aqueous innature. For example, in certain embodiments, a suitable vehicle orcarrier can be water for injection, physiological saline solution orartificial cerebrospinal fluid, possibly supplemented with othermaterials common in compositions for parenteral administration. In someembodiments, the saline comprises isotonic phosphate-buffered saline. Incertain embodiments, neutral buffered saline or saline mixed with serumalbumin are further exemplary vehicles. In certain embodiments,pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, oracetate buffer of about pH 4.0-5.5, which can further include sorbitolor a suitable substitute therefore. In certain embodiments, acomposition comprising an antigen binding protein to PCSK9, with orwithout at least one additional therapeutic agents, can be prepared forstorage by mixing the selected composition having the desired degree ofpurity with optional formulation agents (Remington's PharmaceuticalSciences, supra) in the form of a lyophilized cake or an aqueoussolution. Further, in certain embodiments, a composition comprising anantigen binding protein to PCSK9, with or without at least oneadditional therapeutic agents, can be formulated as a lyophilizate usingappropriate excipients such as sucrose.

In certain embodiments, the pharmaceutical composition can be selectedfor parenteral delivery. In certain embodiments, the compositions can beselected for inhalation or for delivery through the digestive tract,such as orally. The preparation of such pharmaceutically acceptablecompositions is within the ability of one skilled in the art.

In certain embodiments, the formulation components are present inconcentrations that are acceptable to the site of administration. Incertain embodiments, buffers are used to maintain the composition atphysiological pH or at a slightly lower pH, typically within a pH rangeof from about 5 to about 8.

In certain embodiments, when parenteral administration is contemplated,a therapeutic composition can be in the form of a pyrogen-free,parenterally acceptable aqueous solution comprising a desired antigenbinding protein to PCSK9, with or without additional therapeutic agents,in a pharmaceutically acceptable vehicle. In certain embodiments, avehicle for parenteral injection is sterile distilled water in which anantigen binding protein to PCSK9, with or without at least oneadditional therapeutic agent, is formulated as a sterile, isotonicsolution, properly preserved. In certain embodiments, the preparationcan involve the formulation of the desired molecule with an agent, suchas injectable microspheres, bio-erodible particles, polymeric compounds(such as polylactic acid or polyglycolic acid), beads or liposomes, thatcan provide for the controlled or sustained release of the product whichcan then be delivered via a depot injection. In certain embodiments,hyaluronic acid can also be used, and can have the effect of promotingsustained duration in the circulation. In certain embodiments,implantable drug delivery devices can be used to introduce the desiredmolecule.

In certain embodiments, a pharmaceutical composition can be formulatedfor inhalation. In certain embodiments, an antigen binding protein toPCSK9, with or without at least one additional therapeutic agent, can beformulated as a dry powder for inhalation. In certain embodiments, aninhalation solution comprising an antigen binding protein to PCSK9, withor without at least one additional therapeutic agent, can be formulatedwith a propellant for aerosol delivery. In certain embodiments,solutions can be nebulized. Pulmonary administration is furtherdescribed in PCT application no. PCT/US94/001875, which describespulmonary delivery of chemically modified proteins.

In certain embodiments, it is contemplated that formulations can beadministered orally. In certain embodiments, an antigen binding proteinto PCSK9, with or without at least one additional therapeutic agents,that is administered in this fashion can be formulated with or withoutthose carriers customarily used in the compounding of solid dosage formssuch as tablets and capsules. In certain embodiments, a capsule can bedesigned to release the active portion of the formulation at the pointin the gastrointestinal tract when bioavailability is maximized andpre-systemic degradation is minimized. In certain embodiments, at leastone additional agent can be included to facilitate absorption of anantigen binding protein to PCSK9 and/or any additional therapeuticagents. In certain embodiments, diluents, flavorings, low melting pointwaxes, vegetable oils, lubricants, suspending agents, tabletdisintegrating agents, and binders can also be employed.

In certain embodiments, a pharmaceutical composition can involve aneffective quantity of an antigen binding protein to PCSK9, with orwithout at least one additional therapeutic agents, in a mixture withnon-toxic excipients which are suitable for the manufacture of tablets.In certain embodiments, by dissolving the tablets in sterile water, oranother appropriate vehicle, solutions can be prepared in unit-doseform. In certain embodiments, suitable excipients include, but are notlimited to, inert diluents, such as calcium carbonate, sodium carbonateor bicarbonate, lactose, or calcium phosphate; or binding agents, suchas starch, gelatin, or acacia; or lubricating agents such as magnesiumstearate, stearic acid, or talc.

Additional pharmaceutical compositions will be evident to those skilledin the art, including formulations involving antigen binding proteins toPCSK9, with or without at least one additional therapeutic agent(s), insustained- or controlled-delivery formulations. In certain embodiments,techniques for formulating a variety of other sustained- orcontrolled-delivery means, such as liposome carriers, bio-erodiblemicroparticles or porous beads and depot injections, are also known tothose skilled in the art. See for example, PCT Application No.PCT/US93/00829 which describes the controlled release of porouspolymeric microparticles for the delivery of pharmaceuticalcompositions. In certain embodiments, sustained-release preparations caninclude semipermeable polymer matrices in the form of shaped articles,e.g. films, or microcapsules. Sustained release matrices can includepolyesters, hydrogels, polylactides (U.S. Pat. No. 3,773,919 and EP058,481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate(Sidman et al., Biopolymers, 22:547-556 (1983)),poly(2-hydroxyethyl-methacrylate) (Langer et al., J. Biomed. Mater.Res., 15:167-277 (1981) and Langer, Chem. Tech., 12:98-105 (1982)),ethylene vinyl acetate (Langer et al., supra) orpoly-D(−)-3-hydroxybutyric acid (EP 133,988). In certain embodiments,sustained release compositions can also include liposomes, which can beprepared by any of several methods known in the art. See, e.g., Eppsteinet al., Proc. Natl. Acad. Sci. USA, 82:3688-3692 (1985); EP 036,676; EP088,046 and EP 143,949.

The pharmaceutical composition to be used for in vivo administrationtypically is sterile. In certain embodiments, this can be accomplishedby filtration through sterile filtration membranes. In certainembodiments, where the composition is lyophilized, sterilization usingthis method can be conducted either prior to or following lyophilizationand reconstitution. In certain embodiments, the composition forparenteral administration can be stored in lyophilized form or in asolution. In certain embodiments, parenteral compositions generally areplaced into a container having a sterile access port, for example, anintravenous solution bag or vial having a stopper pierceable by ahypodermic injection needle.

In certain embodiments, once the pharmaceutical composition has beenformulated, it can be stored in sterile vials as a solution, suspension,gel, emulsion, solid, or as a dehydrated or lyophilized powder. Incertain embodiments, such formulations can be stored either in aready-to-use form or in a form (e.g., lyophilized) that is reconstitutedprior to administration.

In certain embodiments, kits are provided for producing a single-doseadministration unit. In certain embodiments, the kit can contain both afirst container having a dried protein and a second container having anaqueous formulation. In certain embodiments, kits containing single andmulti-chambered pre-filled syringes (e.g., liquid syringes andlyosyringes) are included.

In certain embodiments, the effective amount of a pharmaceuticalcomposition comprising an antigen binding protein to PCSK9, with orwithout at least one additional therapeutic agent, to be employedtherapeutically will depend, for example, upon the therapeutic contextand objectives. One skilled in the art will appreciate that theappropriate dosage levels for treatment, according to certainembodiments, will thus vary depending, in part, upon the moleculedelivered, the indication for which an antigen binding protein to PCSK9,with or without at least one additional therapeutic agent, is beingused, the route of administration, and the size (body weight, bodysurface or organ size) and/or condition (the age and general health) ofthe patient. In certain embodiments, the clinician can titer the dosageand modify the route of administration to obtain the optimal therapeuticeffect. In certain embodiments, a typical dosage can range from about0.1 μg/kg to up to about 100 mg/kg or more, depending on the factorsmentioned above. In certain embodiments, the dosage can range from 0.1μg/kg up to about 100 mg/kg; or 1 μg/kg up to about 100 mg/kg; or 5μg/kg up to about 100 mg/kg.

In certain embodiments, the frequency of dosing will take into accountthe pharmacokinetic parameters of an antigen binding protein to PCSK9and/or any additional therapeutic agents in the formulation used. Incertain embodiments, a clinician will administer the composition until adosage is reached that achieves the desired effect. In certainembodiments, the composition can therefore be administered as a singledose, or as two or more doses (which may or may not contain the sameamount of the desired molecule) over time, or as a continuous infusionvia an implantation device or catheter. Further refinement of theappropriate dosage is routinely made by those of ordinary skill in theart and is within the ambit of tasks routinely performed by them. Incertain embodiments, appropriate dosages can be ascertained through useof appropriate dose-response data. In some embodiments, the amount andfrequency of administration can take into account the desiredcholesterol level (serum and/or total) to be obtained and the subject'spresent cholesterol level, LDL level, and/or LDLR levels, all of whichcan be obtained by methods that are well known to those of skill in theart.

In certain embodiments, the route of administration of thepharmaceutical composition is in accord with known methods, e.g. orally,through injection by intravenous, intraperitoneal, intracerebral(intra-parenchymal), intracerebroventricular, intramuscular,subcutaneously, intra-ocular, intraarterial, intraportal, orintralesional routes; by sustained release systems or by implantationdevices. In certain embodiments, the compositions can be administered bybolus injection or continuously by infusion, or by implantation device.

In certain embodiments, the composition can be administered locally viaimplantation of a membrane, sponge or another appropriate material ontowhich the desired molecule has been absorbed or encapsulated. In certainembodiments, where an implantation device is used, the device can beimplanted into any suitable tissue or organ, and delivery of the desiredmolecule can be via diffusion, timed-release bolus, or continuousadministration.

In certain embodiments, it can be desirable to use a pharmaceuticalcomposition comprising an antigen binding protein to PCSK9, with orwithout at least one additional therapeutic agent, in an ex vivo manner.In such instances, cells, tissues and/or organs that have been removedfrom the patient are exposed to a pharmaceutical composition comprisingan antigen binding protein to PCSK9, with or without at least oneadditional therapeutic agent, after which the cells, tissues and/ororgans are subsequently implanted back into the patient.

In certain embodiments, an antigen binding protein to PCSK9 and/or anyadditional therapeutic agents can be delivered by implanting certaincells that have been genetically engineered, using methods such as thosedescribed herein, to express and secrete the polypeptides. In certainembodiments, such cells can be animal or human cells, and can beautologous, heterologous, or xenogeneic. In certain embodiments, thecells can be immortalized. In certain embodiments, in order to decreasethe chance of an immunological response, the cells can be encapsulatedto avoid infiltration of surrounding tissues. In certain embodiments,the encapsulation materials are typically biocompatible, semi-permeablepolymeric enclosures or membranes that allow the release of the proteinproduct(s) but prevent the destruction of the cells by the patient'simmune system or by other detrimental factors from the surroundingtissues.

Based on the ability of ABPs to significantly neutralize PCSK9 activity(as demonstrated in the Examples below), these ABPs will havetherapeutic effects in treating and preventing symptoms and conditionsresulting from PCSK9-mediated activity, such as hypercholesterolemia.

Diagnostic Applications

In some embodiments, the ABP is used as a diagnostic tool. The ABP canbe used to assay the amount of PCSK9 present in a sample and/or subject.As will be appreciated by one of skill in the art, such ABPs need not beneutralizing ABPs. In some embodiments, the diagnostic ABP is not aneutralizing ABP. In some embodiments, the diagnostic ABP binds to adifferent epitope than the neutralizing ABP binds to. In someembodiments, the two ABPs do not compete with one another.

In some embodiments, the ABPs disclosed herein are used or provided inan assay kit and/or method for the detection of PCSK9 in mammaliantissues or cells in order to screen/diagnose for a disease or disorderassociated with changes in levels of PCSK9. The kit comprises an ABPthat binds PCSK9 and means for indicating the binding of the ABP withPCSK9, if present, and optionally PCSK9 protein levels. Various meansfor indicating the presence of an ABP can be used. For example,fluorophores, other molecular probes, or enzymes can be linked to theABP and the presence of the ABP can be observed in a variety of ways.The method for screening for such disorders can involve the use of thekit, or simply the use of one of the disclosed ABPs and thedetermination of whether the ABP binds to PCSK9 in a sample. As will beappreciated by one of skill in the art, high or elevated levels of PCSK9will result in larger amounts of the ABP binding to PCSK9 in the sample.Thus, degree of ABP binding can be used to determine how much PCSK9 isin a sample. Subjects or samples with an amount of PCSK9 that is greaterthan a predetermined amount (e.g., an amount or range that a personwithout a PCSK9 related disorder would have) can be characterized ashaving a PCSK9 mediated disorder. In some embodiments, the ABP isadministered to a subject taking a statin, in order to determine if thestatin has increased the amount of PCSK9 in the subject.

In some embodiments, the ABP is a non-neutralizing ABP and is used todetermine the amount of PCSK9 in a subject receiving an ABP and/orstatin treatment.

EXAMPLES

The following examples, including the experiments conducted and resultsachieved, are provided for illustrative purposes only and are not to beconstrued as limiting the present invention.

Example 1 Immunization and Titering Generation of Anti-PCSK9 Antibodiesand Hybridomas

Antibodies to the mature form of PCSK9 (depicted as the sequence in FIG.1A, with the pro-domain underlined), were raised in XenoMouse® mice(Abgenix, Fremont, Calif.), which are mice containing humanimmunoglobulin genes. Two groups of XenoMouse® mice, group 1 and 2, wereused to produce antibodies to PCSK9. Group 1 included mice of theXenoMouse® strain XMG2-KL, which produces fully human IgG2_(κ) and IgG2λantibodies. Group 1 mice were immunized with human PCSK9. PCSK9 wasprepared using standard recombinant techniques using the GenBanksequence as reference (NM_(—)174936). Group 2 involved mice of theXenoMouse®strain XMG4-KL, which produce fully human IgG4_(κ) and IgG4λantibodies. Group 2 mice were also immunized with human PCSK9.

The mice of both groups were injected with antigen eleven times,according to the schedule in Table 3. In the initial immunizations, eachmouse was injected with a total of 10 μg of antigen deliveredintraperitoneally into the abdomen. Subsequent boosts are 5 ug doses andinjection method is staggered between intraperitoneal injections intothe abdomen and sub-cutaneous injections at the base of the tail. Forintraperitoneal injections antigen is prepared as an emulsion withTiterMax® Gold (Sigma, Cat # T2684) and for subcutaneous injectionsantigen is mixed with Alum (aluminum phosphate) and CpG oligos. Ininjections 2 through 8 and 10, each mouse was injected with a total of 5μg of antigen in the adjuvant alum gel. A final injection of 5 μg ofantigen per mouse is delivered in Phospho buffered saline and deliveredinto 2 sites 50% IP into the abdomen and 50% SQ at the base of tail. Theimmunization programs are summarized in Table 3, shown below.

TABLE 3 mouse strain XMG2/kl XMG4/kl # of animals 10 10 immunogen  1stboost PCSK9-V5/His PCSK9-V5/His IP injection IP injection 10 ug each 10ug each Titermax Gold Titermax Gold  2nd boost tail injection tailinjection 5 ug each 5 ug each Alum/CpG ODN Alum/CpG ODN  3rd boost IPinjection IP injection 5 ug each 5 ug each Titermax Gold Titermax Gold 4th boost tail injection tail injection 5 ug each 5 ug each Alum/CpGODN Alum/CpG ODN  5th boost IP injection IP injection 5 ug each 5 ugeach Titermax Gold Titermax Gold  6th boost tail injection tailinjection 5 ug each 5 ug each Alum/CpG ODN Alum/CpG ODN  7th boost IPinjection IP injection 5 ug each 5 ug each Titermax Gold Titermax Gold 8th boost tail injection tail injection 5 ug each 5 ug each Alum/CpGODN Alum/CpG ODN bleed  9th boost IP injection IP injection 5 ug each 5ug each Titermax Gold Titermax Gold 10th boost tail injection tailinjection 5 ug each 5 ug each Alum/CpG ODN Alum/CpG ODN 11th boost BIPBIP 5 ug each 5 ug each PBS PBS harvest

The protocol used to titer the XenoMouse animals was as follows: Costar3368 medium binding plates were coated with neutravadin @ 8 ug/ml (50ul/well) and incubated at 4° C. in 1×PBS/0.05% azide overnight. Theywere washed using TiterTek 3-cycle wash with RO water. Plates wereblocked using 250 ul of 1×PBS/1% milk and incubated for at least 30minutes at RT. Block was washed off using TiterTek 3-cycle wash with ROwater. One then captured b-human PCSK9 @ 2 ug/ml in 1×PBS/1% milk/10 mMCa2+ (assay diluent) 50 ul/well and incubated for 1 hr at RT. One thenwashed using TiterTek 3-cycle wash with RO water. For the primaryantibody, sera was titrated 1:3 in duplicate from 1:100. This was donein assay diluent 50 ul/well and incubated for 1 hr at RT. One thenwashed using TiterTek 3-cycle wash with RO water. The secondary antibodywas goat anti Human IgG Fc HRP @ 400 ng/ml in assay diluent at 50ul/well. This was incubated for 1 hr at RT. This was then washed usingTiterTek 3-cycle wash with RO water and patted dry on paper towels. Forthe substrate, one-step TMB solution (Neogen, Lexington, Ky.) was used(50 ul/well) and it was allowed to develop for 30 min at RT.

The protocols followed in the ELISA assays was as follows: For samplescomprising b-PCSK9 with no V5His tag the following protocol wasemployed: Costar 3368 medium binding plates (Corning Life Sciences) wereemployed. The plates were coated with neutravadin at 8 μg/ml in1×PBS/0.05% Azide, (50 μl/well). The plates were incubated at 4° C.overnight. The plates were then washed using a Titertek M384 platewasher (Titertek, Huntsville, Ala.). A 3-cycle wash was performed. Theplates were blocked with 250 μl of 1×PBS/1% milk and incubatedapproximately 30 minutes at room temperature. The plates were thenwashed using the M384 plate washer. A 3-cycle wash was performed. Thecapture was b-hu PCSK9, without a V5 tag, and was added at 2 μg/ml in1×PBS/1% milk/10 mM Ca²⁺ (40 μl/well). The plates were then incubatedfor 1 hour at room temperature. A 3-cycle wash was performed. Sera weretitrated 1:3 in duplicate from 1:100, and row H was blank for sera. Thetitration was done in assay diluent, at a volume of 50 μl/well. Theplates were incubated for 1 hour at room temperature. Next, a 3-cyclewash was performed. Goat anti Human IgG Fc HRP at 100 ng/ml (1:4000) in1×PBS/1% milk/10 mM Ca²⁺ (50 μl/well) was added to the plate and wasincubated 1 hour at room temperature. The plates were washed once again,using a 3-cycle wash. The plates were then patted dry with paper towel.Finally, 1 step TMB (Neogen, Lexington, Ky.) (50 μl/well) was added tothe plate and was quenched with 1N hydrochloric acid (50 μl/well) after30 minutes at room temperature. OD's were read immediately at 450 nmusing a Titertek plate reader.

Positive controls to detect plate bound PCSK9 were soluble LDL receptor(R&D Systems, Cat #2148LD/CF) and a polyclonal rabbit anti-PCSK9antibody (Caymen Chemical #10007185) titrated 1:3 in duplicate from 3μg/ml in assay diluent. LDLR was detected with goat anti LDLR (R&DSystems, Cat #AF2148) and rabbit anti goat IgGFc HRP at a concentrationof 400 ng/ml; the rabbit polyclonal was detected with goat anti-rabbitIgG Fc at a concentration of 400 ng/ml in assay diluent. Negativecontrol was naive XMG2-KL and XMG4-KL sera titrated 1:3 in duplicatefrom 1:100 in assay diluent.

For samples comprising b-PCSK9 with a V5His tag the following protocolwas employed: Costar 3368 medium binding plates (Corning Life Sciences)were employed. The plates were coated with neutravadin at 8 μg/ml in1×PBS/0.05% Azide, (50 μl/well). The plates were incubated at 4° C.overnight. The plates were then washed using a Titertek M384 platewasher (Titertek, Huntsville, Ala.). A 3-cycle wash was performed. Theplates were blocked with 250 μl of 1×PBS/1% milk and incubatedapproximately 30 minutes at room temperature. The plates were thenwashed using the M384 plate washer. A 3-cycle wash was performed. Thecapture was b-hu PCSK9, with a V5 tag, and was added at 2 μg/ml in1×PBS/1% milk/10 mM Ca²⁺ (40 μl/well). The plates were then incubatedfor 1 hour at room temperature. A 3-cycle wash was performed. Sera weretitrated 1:3 in duplicate from 1:100, and row H was blank for sera. Thetitration was done in assay diluent, at a volume of 50 μl/well. Theplates were incubated for 1 hour at room temperature. Next, the plateswere washed using the M384 plate washer operated using a 3-cycle wash.Goat anti Human IgG Fc HRP at 400 ng/ml in 1×PBS/1% milk/10 mM Ca²⁺ wasadded at 50 μl/well to the plate and the plate was incubated 1 hour atroom temperature. The plates were washed once again, using a 3-cyclewash. The plates were then patted dry with paper towel. Finally, 1 stepTMB (Neogen, Lexington, Ky.) (50 μl/well) was added to the plate and theplate was quenched with 1N hydrochloric acid (50 μl/well) after 30minutes at room temperature. OD's were read immediately at 450 nm usinga Titertek plate reader.

Positive control was LDLR, rabbit anti-PCSK9 titrated 1:3 in duplicatefrom 3 μg/ml in assay diluent. LDLR detect with goat anti-LDLR (R&DSystems, Cat #AF2148) and rabbit anti-goat IgG Fc HRP at a concentrationof 400 ng/ml; rabbit poly detected with goat anti-rabbit IgG Fc at aconcentration of 400 ng/ml in assay diluent. Human anti-His 1.2,3 andanti-V5 1.7.1 titrated 1:3 in duplicate from 1 μg/ml in assay diluent;both detected with goat anti-human IgG Fc HRP at a concentration of 400ng/ml in assay diluent. Negative control was naive XMG2-KL and XMG4-KLsera titrated 1:3 in duplicate from 1:100 in assay diluent.

Titers of the antibody against human PCSK9 were tested by ELISA assayfor mice immunized with soluble antigen as described. Table 4 summarizesthe ELISA data and indicates that there were some mice which appeared tobe specific for PCSK9. See, e.g., Table 4. Therefore, at the end of theimmunization program, 10 mice (in bold in Table 4) were selected forharvest, and splenocytes and lymphocytes were isolated from the spleensand lymph nodes respectively, as described herein.

TABLE 4 Summary of ELISA Results Titer Titer Animal b-hu PCSK9 b-huPCSK9 @ ID (V5His) @ 2 ug/ml 2 ug/ml Group 1 - P175807 >72900 @ OD 2.268359 IgG2k/l P175808 >72900 @ OD 2.3 >72900 @ OD 2.5 P175818 >72900 @OD 3.2 >72900 @ OD 3.0 P175819 >72900 @ OD 3.4 >72900 @ OD 3.2P175820 >72900 @ OD 2.4 >72900 @ OD 2.5 P175821 >72900 @ OD 3.4 >72900 @OD 3.0 P175830 >72900 @ OD 2.6 >72900 @ OD 2.5 P175831 >72900 @ OD3.1 >72900 @ OD 3.1 P175832 >72900 @ OD 3.8 >72900 @ OD 3.6P175833 >72900 @ OD 2.6 >72900 @ OD 2.3 Group 2 - P174501 19369 17109IgG4k/l P174503 31616 23548 P174508 48472 30996 P174509 23380 21628P174510 15120  9673 P175773 19407 15973 P175774 54580 44424 P17577560713 55667 P175776 30871 22899 P175777 16068 12532 Naïve <100 @ OD 0.54<100 @ OD 0.48 G2 Naïve <100 @ OD 1.57 <100 @ OD 1.32 G4

Example 2 Recovery of Lymphocytes, B-Cell Isolations, Fusions andGeneration of Hybridomas

This example outlines how the immune cells were recovered and thehybridomas were generated. Selected immunized mice were sacrificed bycervical dislocation and the draining lymph nodes were harvested andpooled from each cohort. The B cells were dissociated from lymphoidtissue by grinding in DMEM to release the cells from the tissues, andthe cells were suspended in DMEM. The cells were counted, and 0.9 mlDMEM per 100 million lymphocytes was added to the cell pellet toresuspend the cells gently but completely.

Lymphocytes were mixed with nonsecretory myeloma P3X63Ag8.653 cellspurchased from ATCC, cat.# CRL 1580 (Kearney et al., (1979) J. Immunol.123, 1548-1550) at a ratio of 1:4. The cell mixture was gently pelletedby centrifugation at 400×g 4 min. After decanting of the supernatant,the cells were gently mixed using a 1 ml pipette. Preheated PEG/DMSOsolution from Sigma (cat# P7306) (1 ml per million of B-cells) wasslowly added with gentle agitation over 1 min followed by 1 min ofmixing. Preheated IDMEM (2 ml per million of B cells) (DMEM withoutglutamine, L-glutamine, pen/strep, MEM non-essential amino acids (allfrom Invitrogen), was then added over 2 minutes with gentle agitation.Finally preheated IDMEM (8 ml per 10⁶ B-cells) was added over 3 minutes.

The fused cells were spun down 400×g 6 min and resuspended in 20 mlselection media (DMEM (Invitrogen), 15 FBS (Hyclone), supplemented withL-glutamine, pen/strep, MEM Non-essential amino acids, Sodium Pyruvate,2-Mercaptoethanol (all from Invitrogen), HA-Azaserine Hypoxanthine andOPI (oxaloacetate, pyruvate, bovine insulin) (both from Sigma) and IL-6(Boehringer Mannheim)) per million B-cells. Cells were incubated for20-30 min at 37C and then resuspended in 200 ml selection media andcultured for 3-4 days in T175 flask prior to 96 well plating. Thus,hybridomas that produced antigen binding proteins to PCSK9 wereproduced.

Example 3 Selection of PCSK9 Antibodies

The present example outlines how the various PCSK9 antigen bindingproteins were characterized and selected. The binding of secretedantibodies (produced from the hybridomas produced in Examples 1 and 2)to PCSK9 was assessed. Selection of antibodies was based on binding dataand inhibition of PCSK9 binding to LDLR and affinity. Binding to solublePCSK9 was analyzed by ELISA, as described below. BIAcore® (surfaceplasmon resonance) was used to quantify binding affinity.

Primary Screen

A primary screen for antibodies which bind to wild-type PCSK9 wasperformed. The primary screen was performed on two harvests. The primaryscreen comprised an ELISA assay and was performed using the followingprotocol:

Costar 3702 medium binding 384 well plates (Corning Life Sciences) wereemployed. The plates were coated with neutravadin at a concentration of4 μg/ml in 1×PBS/0.05% Azide, at a volume of 40 μl/well. The plates wereincubated at 4° C. overnight. The plates were then washed using aTitertek plate washer (Titertek, Huntsville, Ala.). A 3-cycle wash wasperformed. The plates were blocked with 90 μl of 1×PBS/1% milk andincubated approximately 30 minutes at room temperature. The plates werethen washed. Again, a 3-cycle wash was performed. The capture sample wasbiotinylated-PCSK9, without a V5 tag, and was added at 0.9 μg/ml in1×PBS/1% milk/10 mM Ca²⁺ at a volume of 40 μl/well. The plates were thenincubated for 1 hour at room temperature. Next, the plates were washedusing the Titertek plate washer operated using a 3-cycle wash. 10 μl ofsupernatant was transferred into 40 μl of 1×PBS/1% milk/10 mM Ca²⁺ andincubated 1.5 hours at room temperature. Again the plates were washedusing the Titertek plate washer operated using a 3-cycle wash. 40μl/well of Goat anti-Human IgG Fc POD at a concentration of 100 ng/ml(1:4000) in 1×PBS/1% milk/10 mM Ca²⁺ was added to the plate and wasincubated 1 hour at room temperature. The plates were washed once again,using a 3-cycle wash. Finally, 40 μl/well of One-step TMB (Neogen,Lexington, Ky.) was added to the plate and quenching with 40 μl/well of1N hydrochloric acid was performed after 30 minutes at room temperature.OD's were read immediately at 450 nm using a Titertek plate reader.

The primary screen resulted in a total of 3104 antigen specifichybridomas being identified from the two harvests. Based on highestELISA OD, 1500 hybridomas per harvest were advanced for a total of 3000positives.

Confirmatory Screen

The 3000 positives were then rescreened for binding to wild-type PCSK9to confirm stable hybridomas were established. The screen was performedas follows: Costar 3702 medium binding 384 well plates (Corning LifeSciences) were employed. The plates were coated with neutravadin at 3μg/ml in 1×PBS/0.05% Azide at a volume of 40 μl/well. The plates wereincubated at 4° C. overnight. The plates were then washed using aTitertek plate washer (Titertek, Huntsville, Ala.). A 3-cycle wash wasperformed. The plates were blocked with 90 μl of 1×PBS/1% milk andincubated approximately 30 minutes at room temperature. The plates werethen washed using the M384 plate washer. A 3-cycle wash was performed.The capture sample was b-PCSK9, without a V5 tag, and was added at 0.9μg/ml in 1×PBS/1% milk/10 mM Ca²⁺ at a volume of 40 μl/well. The plateswere then incubated for 1 hour at room temperature. Next, the plateswere washed using a 3-cycle wash. 10 μl of supernatant was transferredinto 40 μl of 1×PBS/1% milk/10 mM Ca²⁺ and incubated 1.5 hours at roomtemperature. Again the plates were washed using the Titertek platewasher operated using a 3-cycle wash. 40 μl/well of Goat anti-Human IgGFc POD at a concentration of 100 ng/ml (1:4000) in 1×PBS/1% milk/10 mMCa²⁺ was added to the plate, and the plate was incubated 1 hour at roomtemperature. The plates were washed once again, using the Titertek platewasher operated using a 3-cycle wash. Finally, 40 μl/well of One-stepTMB (Neogen, Lexington, Ky.) was added to the plate and was quenchedwith 40 μl/well of 1N hydrochloric acid after 30 minutes at roomtemperature. OD's were read immediately at 450 nm using a Titertek platereader. A total of 2441 positives repeated in the second screen. Theseantibodies were then used in the subsequent screenings.

Mouse Cross-Reactivity Screen

The panel of hybridomas was then screened for cross-reactivity to mousePCSK9 to make certain that the antibodies could bind to both human andmouse PCSK9. The following protocol was employed in the cross-reactivityscreen: Costar 3702 medium binding 384 well plates (Corning LifeSciences) were employed. The plates were coated with neutravadin at 3μg/ml in 1×PBS/0.05% Azide at a volume of 40 μl/well. The plates wereincubated at 4° C. overnight. The plates were then washed using aTitertek plate washer (Titertek, Huntsville, Ala.). A 3-cycle wash wasperformed. The plates were blocked with 90 μl of 1×PBS/1% milk andincubated approximately 30 minutes at room temperature. The plates werethen washed using the Titertek plate washer. A 3-cycle wash wasperformed. The capture sample was biotinylated-mouse PCSK9, and wasadded at 1 μg/ml in 1×PBS/1% milk/10 mM Ca²⁺ at a volume of 40 μl/well.The plates were then incubated for 1 hour at room temperature. Next, theplates were washed using the Titertek plate washer operated using a3-cycle wash. 50 μl of supernatant was transferred to the plates andincubated 1 hour at room temperature. Again the plates were washed usinga 3-cycle wash. 40 μl/well of Goat anti-Human IgG Fc POD at aconcentration of 100 ng/ml (1:4000) in 1×PBS/1% milk/10 mM Ca²⁺ wasadded to the plate and the plate was incubated 1 hour at roomtemperature. The plates were washed once again, using a 3-cycle wash.Finally, 40 μl/well One-step TMB (Neogen, Lexington, Ky.) was added tothe plate and was quenched with 40 μl/well of 1N hydrochloric acid after30 minutes at room temperature. OD's were read immediately at 450 nmusing a Titertek plate reader. 579 antibodies were observed tocross-react with mouse PCSK9. These antibodies were then used in thesubsequent screenings.

D374Y Mutant Binding Screen

The D374Y mutation in PCSK9 has been documented in the human population(e.g., Timms K M et al, “A mutation in PCSK9 causing autosomal-dominanthypercholesterolemia in a Utah pedigree”, Hum. Genet. 114: 349-353,2004). In order to determine if the antibodies were specific for thewild type or also bound to the D374Y form of PCSK9, the samples werethen screened for binding to the mutant PCSK9 sequence comprising themutation D374Y. The protocol for the screen was as follows: Costar 3702medium binding 384 well plates (Corning Life Sciences) were employed inthe screen. The plates were coated with neutravadin at 4 μg/ml in1×PBS/0.05% Azide at a volume of 40 μl/well. The plates were incubatedat 4° C. overnight. The plates were then washed using a Titertek platewasher (Titertek, Huntsville, Ala.). A 3-cycle wash was performed. Theplates were blocked with 90 μl of 1×PBS/1% milk and incubatedapproximately 30 minutes at room temperature. The plates were thenwashed using the Titertek plate washer. A 3-cycle wash was performed.The plates were coated with biotinylated human PCSK9 D374Y at aconcentration of 1 μg/ml in 1×PBS/1% milk/10 mMCa²⁺ and incubated for 1hour at room temperature. The plates were then washed using a Titertekplate washer. A 3-cycle wash was performed. Late exhaust hybridomaculture supernatant was diluted 1:5 in PBS/milk/Ca²⁺ (10 ml plus 40 ml)and incubated for 1 hour at room temperature. Next, 40 μl/well of rabbitanti-human PCSK9 (Cayman Chemical) and human anti-His 1.2.3 1:2 at 1ug/ml in 1×PBS/1% milk/10 mMCa²⁺ was titrated onto the plates, whichwere then incubated for 1 hour at room temperature. The plates were thenwashed using a Titertek plate washer. A 3-cycle wash was performed. 40μl/well of Goat anti-Human IgG Fc HRP at a concentration of 100 ng/ml(1:4000) in 1×PBS/1% milk/10 mM Ca²⁺ was added to the plate and theplate was incubated 1 hour at room temperature. 40 μl/well of Goatanti-rabbit IgG Fc HRP at a concentration of 100 ng/ml (1:4000) in1×PBS/1% milk/10 mM Ca²⁺ was added to the plate and the plate wasincubated 1 hour at room temperature. The plates were then washed usinga Titertek plate washer. A 3-cycle wash was performed. Finally, 40μl/well of One-step TMB (Neogen, Lexington, Ky.) was added to the plateand was quenched with 40 μl/well of 1N hydrochloric acid after 30minutes at room temperature. OD's were read immediately at 450 nm usinga Titertek plate reader. Over 96% of the positive hits on the wild-typePCSK9 also bound mutant PCSK9.

Large Scale Receptor Ligand Blocking Screen

To screen for the antibodies that block PCSK9 binding to LDLR an assaywas developed using the D374Y PCSK9 mutant. The mutant was used for thisassay because it has a higher binding affinity to LDLR allowing a moresensitive receptor ligand blocking assay to be developed. The followingprotocol was employed in the receptor ligand blocking screen: Costar3702 medium binding 384 well plates (Corning Life Sciences) wereemployed in the screen. The plates were coated with goat anti-LDLR (R&DCat #AF2148) at 2 μg/ml in 1×PBS/0.05% Azide at a volume of 40 μl/well.The plates were incubated at 4° C. overnight. The plates were thenwashed using a Titertek plate washer (Titertek, Huntsville, Ala.). A3-cycle wash was performed. The plates were blocked with 90 μl of1×PBS/1% milk and incubated approximately 30 minutes at roomtemperature. The plates were then washed using the Titertek platewasher. A 3-cycle wash was performed. The capture sample was LDLR (R&D,Cat #2148LD/CF), and was added at 0.4 μg/ml in 1×PBS/1% milk/10 mM Ca²⁺at a volume of 40 μl/well. The plates were then incubated for 1 hour and10 minutes at room temperature. Contemporaneously, 20 ng/ml ofbiotinylated human D374Y PCSK9 was incubated with 15 microliters ofhybridoma exhaust supernatant in Nunc polypropylene plates and theexhaust supernatant concentration was diluted 1:5. The plates were thenpre-incubated for about 1 hour and 30 minutes at room temperature. Next,the plates were washed using the Titertek plate washer operated using a3-cycle wash. 50 μl/well of the pre-incubated mixture was transferredonto the LDLR coated ELISA plates and incubated for 1 hour at roomtemperature. To detect LDLR-bound b-PCSK9, 40 μl/well streptavidin HRPat 500 ng/ml in assay diluent was added to the plates. The plates wereincubated for 1 hour at room temperature. The plates were again washedusing a Titertek plate washer. A 3-cycle wash was performed. Finally, 4μl/well of One-step TMB (Neogen, Lexington, Ky.) was added to the plateand was quenched with 40 μl/well of 1N hydrochloric acid after 30minutes at room temperature. OD's were read immediately at 450 nm usinga Titertek plate reader. The screen identified 384 antibodies thatblocked the interaction between PCSK9 and the LDLR well, 100 antibodiesblocked the interaction strongly (OD<0.3). These antibodies inhibitedthe binding interaction of PCSK9 and LDLR greater than 90% (greater than90% inhibition).

Receptor for Ligand and Binding Assay on Blocker Subset

The receptor ligand assay was then repeated using the mutant enzyme onthe 384 member subset of neutralizers identified in the first largescale receptor ligand inhibition assay. The same protocol was employedin the screen of the 384 member blocker subset assay as was done in thelarge scale receptor ligand blocking screen. This repeat screenconfirmed the initial screening data.

This screen of the 384 member subset identified 85 antibodies thatblocked interaction between the PCSK9 mutant enzyme and the LDLR greaterthan 90%.

Receptor Ligand and Binding Assay of Blockers that Bind the Wild TypePCSK9 but not the D374Y Mutant

In the initial panel of 3000 sups there were 86 antibodies shown tospecifically bind to the wild-type PCSK9 and not to the huPCSK9(D374Y)mutant. These 86 sups were tested for the ability to block wild-typePCSK9 binding to the LDLR receptor. The following protocol was employed:Costar 3702 medium binding 384 well plates (Corning Life Sciences) wereemployed in the screen. The plates were coated with anti-His 1.2.3 at 10μg/ml in 1×PBS/0.05% Azide at a volume of 40 μl/well. The plates wereincubated at 4° C. overnight. The plates were then washed using aTitertek plate washer (Titertek, Huntsville, Ala.). A 3-cycle wash wasperformed. The plates were blocked with 90 μl of 1×PBS/1% milk andincubated approximately 30 minutes at room temperature. The plates werethen washed using the Titertek plate washer. A 3-cycle wash wasperformed. LDLR (R&D Systems, #2148LD/CF or R&D Systems, #2148LD) wasadded at 5 μg/ml in 1×PBS/1% milk/10 mM Ca²⁺ at a volume of 40 μl/well.The plates were then incubated for 1 hour at room temperature. Next, theplates were washed using the Titertek plate washer operated using a3-cycle wash. Contemporaneously, biotinylated human wild-type PCSK9 waspre-incubated with hybridoma exhaust supernatant in Nunc polypropyleneplates. 22 μl of hybridoma sup was transferred into 33 ul of b-PCSK9 ata concentration of 583 ng/ml in 1×PBS/1% milk/10 mMCa2+, giving a finalb-PCSK9 concentration=350 ng/ml and the exhaust supernatant at a finaldilution of 1:2.5. The plates were pre-incubated for approximately 1hour and 30 minutes at room temperature. 50 μl/well of the preincubatedmixture was transferred onto LDLR captured ELISA plates and incubatedfor 1 hour at room temperature. The plates were then washed using theTitertek plate washer. A 3-cycle wash was performed. 40 μl/wellstreptavidin HRP at 500 ng/ml in assay diluent was added to the plates.The plates were incubated for 1 hour at room temperature. The plateswere then washed using a Titertek plate washer. A 3-cycle wash wasperformed. Finally, 40 μl/well of One-step TMB (Neogen, Lexington, Ky.)was added to the plate and was quenched with 40 μl/well of 1Nhydrochloric acid after 30 minutes at room temperature. OD's were readimmediately at 450 nm using a Titertek plate reader.

Screening Results

Based on the results of the assays described, several hybridoma lineswere identified as producing antibodies with desired interactions withPCSK9. Limiting dilution was used to isolate a manageable number ofclones from each line. The clones were designated by hybridoma linenumber (e.g. 21B12) and clone number (e.g. 21B12.1). In general, nodifference among the different clones of a particular line were detectedby the functional assays described herein. In a few cases, clones wereidentified from a particular line that behaved differently in thefunctional assays, for example, 25A7.1 was found not to block PCSK9/LDLRbut 25A7.3 (referred to herein as 25A7) was neutralizing. The isolatedclones were each expanded in 50-100 ml of hybridoma media and allowed togrow to exhaustion, (i.e., less than about 10% cell viability). Theconcentration and potency of the antibodies to PCSK9 in the supernatantsof those cultures were determined by ELISA and by in vitro functionaltesting, as described herein. As a result of the screening describedherein, the hybridomas with the highest titer of antibodies to PCSK9were identified. The selected hybridomas are shown in FIGS. 2A-3D andTable 2.

Example 4.1 Production of Human 31H4 IgG4 Antibodies from Hybridomas

This example generally describes how one of the antigen binding proteinswas produced from a hybridoma line. The production work used 50 mlexhaust supernatant generation followed by protein A purification.Integra production was for scale up and was performed later. Hybridomaline 31H4 was grown in T75 flasks in 20 ml of media (Integra Media,Table 5). When the hybridoma was nearly confluent in the T75 flasks, itwas transferred to an Integra flask (Integra Biosciences, IntegraCL1000, cat#90 005).

The Integra flask is a cell culture flask that is divided by a membraneinto two chambers, a small chamber and a large chamber. A volume of20-30 ml hybridoma cells at a minimum cell density of 1×10⁶ cells per mlfrom the 31H4 hybridoma line was placed into the small chamber of anIntegra flask in Integra media (see Table 5 for components of Integramedia). Integra media alone (1 L) was placed in the large chambers ofthe Integra flasks. The membrane separating the two chambers ispermeable to small molecular weight nutrients but is impermeable tohybridoma cells and to antibodies produced by those cells. Thus, thehybridoma cells and the antibodies produced by those hybridoma cellswere retained in the small chamber.

After one week, media was removed from both chambers of the Integraflask and was replaced with fresh Integra media. The collected mediafrom the small chambers was separately retained. After a second week ofgrowth, the media from the small chamber was again collected. Thecollected media from week 1 from the hybridoma line was combined withthe collected media from week 2 from the hybridoma line. The resultingcollected media sample from the hybridoma line was spun to remove cellsand debris (15 minutes at 3000 rpm) and the resulting supernatant wasfiltered (0.22 um). Clarified conditioned media was loaded onto aProtein A-Sepharose column. Optionally, the media can be firstconcentrated and then loaded onto a Protein A Sepharose column.Non-specific bindings were removed by an extensive PBS wash. Boundantibody proteins on the Protein A column were recovered by standardacidic antibody elution from Protein A columns (such as 50 mM Citrate,pH 3.0). Aggregated antibody proteins in the Protein A Sepharose poolwere removed by size exclusion chromatography or binding ion exchangechromatography on anion exchanger resin such as Q Sepharose resin. Thespecific IEX conditions for the 31H4 proteins are Q-Sepharose HP at pH7.8-8.0. Antibody was eluted with a NaCl gradient of 10 mM-500 mM in 25column volumes.

TABLE 5 Composition of Media INTEGRA MEDIA HSFM 10% Ultra Low IgG serum2 mmol/L L-glutamine  1% NEAA 4 g/L glucose

Example 4.2 Production of Recombinant 31H4 Human IgG2 Antibodies fromTransfected Cells

The present example outlines how 31H4 IgG2 antibodies were produced fromtransfected cells. 293 cells for transient expression and CHO cells forstable expression were transfected with plasmids that encode 31H4 heavyand light chains. Conditioned media from transfected cells was recoveredby removing cells and cell debris. Clarified conditioned media wasloaded onto a Protein A-Sepharose column. Optionally, the media canfirst be concentrated and then loaded onto a Protein A Sepharose column.Non-specific bindings were removed by extensive PBS wash. Bound antibodyproteins on the Protein A column were recovered by standard acidicantibody elution from Protein A columns (such as 50 mM citrate, pH 3.0).Aggregated antibody proteins in the Protein A Sepharose pool wereremoved by size exclusion chromatography or binding ion exchangechromatography on anion exchanger resin such as Q Sepharose resin. Thespecific IEX conditions for the 31H4 proteins are Q-Sepharose HP at pH7.8-8.0. The antibody was eluted with a NaCl gradient of 10 mM-500 mM in25 column volumes.

Example 5 Production of Human 21B12 IgG4 Antibodies from Hybridomas

The present example outlines how antibody 21B12 IgG4 was produced fromhybridomas. Hybridoma line 21B12 was grown in T75 flasks in media(Integra Media, Table 5). When the hybridomas were nearly confluent inthe T75 flasks, they were transferred to Integra flasks (IntegraBiosciences, Integra CL1000, cat#90 005).

The Integra flask is a cell culture flask that is divided by a membraneinto two chambers, a small chamber and a large chamber. A volume of20-30 ml hybridoma cells at a minimum cell density of 1×10⁶ cells per mlfrom the 31H4 hybridoma line was placed into the small chamber of anIntegra flask in Integra media (see Table 5 for components of Integramedia). Integra media alone (1 L) was placed in the large chambers ofthe Integra flasks. The membrane separating the two chambers ispermeable to small molecular weight nutrients but is impermeable tohybridoma cells and to antibodies produced by those cells. Thus, thehybridoma cells and the antibodies produced by those hybridoma cellswere retained in the small chamber. After one week, media was removedfrom both chambers of the Integra flask and was replaced with freshIntegra media. The collected media from the small chambers wasseparately retained. After a second week of growth, the media from thesmall chamber was again collected. The collected media from week 1 fromthe hybridoma line was combined with the collected media from week 2from the hybridoma line. The resulting collected media sample from thehybridoma line was spun to remove cells and debris (15 minutes at 3000rpm) and the resulting supernatant was filtered (0.22 μm). Clarifiedconditioned media were loaded onto a Protein A Sepharose column.Optionally, the media are first concentrated and then loaded onto aProtein A Sepharose column. Non-specific bindings were removed by anextensive PBS wash. Bound antibody proteins on the Protein A column wererecovered by standard acidic antibody elution from Protein A columns(such as 50 mM Citrate, pH 3.0). Aggregated antibody proteins in theProtein A Sepharose pool were removed by size exclusion chromatographyor binding ion exchange chromatography on anion exchanger resin such asQ Sepharose resin. The specific IEX conditions for the 21B12 proteinsare Q-Sepharose HP at pH 7.8-8.0. The antibody was eluted with a NaClgradient of 10 mM-500 mM in 25 column volumes.

Example 6 Production of Human 21B12 IgG2 Antibodies from TransfectedCells

The present example outlines how 21B12 IgG2 antibodies were producedfrom transfected cells. Cells (293 cells for transient expression andCHO cells for stable expression) were transfected with plasmids thatencode 21B12 heavy and light chains. Conditioned media from hybridomacells were recovered by removing cells and cell debris. Clarifiedconditioned media were loaded onto a Protein A-Sepharose column.Optionally, the media can first be concentrated and then loaded onto aProtein A Sepharose column. Non-specific bindings were removed byextensive PBS wash. Bound antibody proteins on the Protein A column wererecovered by standard acidic antibody elution from Protein A columns (50mM Citrate, pH 3.0). Aggregated antibody proteins in the Protein ASepharose pool were removed by size exclusion chromatography or bindingion exchange chromatography on cation exchanger resin such asSP-Sepharose resin. The specific IEX conditions for the 21B12 proteinswere SP-Sepharose HP at pH 5.2. Antibodies were eluted with 25 columnvolumes of buffer that contains a NaCl gradient of 10 mM-500 mM in 20 mMsodium acetate buffer.

Example 7 Production of Human 16F12 IgG4 Antibodies from Hybridomas

The present example outlines how antibody 16F12 IgG4 was produced fromhybridomas. Hybridoma line 16F12 was grown in T75 flasks in media (seeTable 5). When the hybridomas were nearly confluent in the T75 flasks,they were transferred to Integra flasks (Integra Biosciences, IntegraCL1000, cat#90 005).

The Integra flask is a cell culture flask that is divided by a membraneinto two chambers, a small chamber and a large chamber. A volume of20-30 ml Hybridoma cells at a minimum cell density of 1×10⁶ cells per mlfrom the 31H4 hybridoma line was placed into the small chamber of anIntegra flask in Integra media (see Table 5 for components of Integramedia). Integra media alone (1 L) was placed in the large chambers ofthe Integra flasks. The membrane separating the two chambers ispermeable to small molecular weight nutrients but is impermeable tohybridoma cells and to antibodies produced by those cells. Thus, thehybridoma cells and the antibodies produced by those hybridoma cellswere retained in the small chamber.

After one week, media was removed from both chambers of the Integraflask and was replaced with fresh Integra media. The collected mediafrom the small chambers was separately retained. After a second week ofgrowth, the media from the small chamber was again collected. Thecollected media from week 1 from the hybridoma line was combined withthe collected media from week 2 from the hybridoma line. The resultingcollected media sample from the hybridoma line were spun to remove cellsand debris (15 minutes at 3000 rpm) and the resulting supernatants werefiltered (0.22 μm). Clarified conditioned media were loaded onto aProtein A Sepharose column. Optionally, the media can be firstconcentrated and then loaded onto a Protein A Sepharose column.Non-specific bindings were removed by extensive PBS wash. Bound antibodyproteins on the Protein A column were recovered by standard acidicantibody elution from Protein A columns (50 mM Citrate, pH 3.0).Aggregated antibody proteins in the Protein A Sepharose pool wereremoved by size exclusion chromatography or binding ion exchangechromatography on anion exchanger resin such as Q Sepharose resin. Thespecific IEX conditions for the 16F12 proteins are Q Sepharose HP at pH7.8-8.0. Antibody was eluted with a NaCl gradient of 10 mM-500 mM in 25column volumes.

Example 8 Production of Human 16F12 IgG2 Antibodies from TransfectedCells

The present example outlines how 16F12 IgG2 antibodies were producedfrom transfected cells. Cells (293 cells for transient expression andCHO cells for stable expression) were transfected with plasmids thatencode 16F12 heavy and light chains. Conditioned media from hybridomacells were recovered by removing cells and cell debris. Clarifiedconditioned media were loaded onto a Protein A-Sepharose. Optionally,the media can be first concentrated and then loaded onto a Protein ASepharose column. Non-specific bindings were removed by extensive PBSwash. Bound antibody proteins on the Protein A column were recovered bystandard acidic antibody elution from Protein A columns (50 mM Citrate,pH 3.0). Aggregated antibody proteins in the Protein A Sepharose poolwere removed by size exclusion chromatography or binding ion exchangechromatography on cation exchanger resin such as SP Sepharose resin. Thespecific IEX conditions for the 16F12 proteins are SP Sepharose HP at pH5.2. Antibody is eluted with 25 column volumes of buffer that contains aNaCl gradient of 10 mM-500 mM in 20 mM sodium acetate buffer.

Example 9 Sequence Analysis of Antibody Heavy and Light Chains

The nucleic acid and amino acid sequences for the light and heavy chainsof the above antibodies were then determined by Sanger (dideoxy)nucleotide sequencing. Amino acid sequences were then deduced for thenucleic acid sequences. The nucleic acid sequences for the variabledomains are depicted in FIGS. 3E-3B.

The cDNA sequences for the lambda light chain variable regions of 31H4,21B12, and 16F12 were determined and are disclosed as SEQ ID NOs: 153,95, and 105 respectively.

The cDNA sequences for the heavy chain variable regions of 31H4, 21B12,and 16F12 were determined and are disclosed as SEQ ID NOs: 152, 94, and104 respectively.

The lambda light chain constant region (SEQ ID NO: 156), and the IgG2and IgG4 heavy chain constant regions (SEQ ID NOs: 154 and 155) areshown in FIG. 3KK.

The polypeptide sequences predicted from each of those cDNA sequenceswere determined. The predicted polypeptide sequences for the lambdalight chain variable regions of 31H4, 21B12, and 16F12 were predictedand are disclosed as SEQ ID NOs: 12, 23, and 35 respectively, the lambdalight chain constant region (SEQ ID NO: 156), the heavy chain variableregions of 31H4, 21B12, and 16F12 were predicted and are disclosed as(SEQ. ID NOs. 67, 49, and 79 respectively. The IgG2 and IgG4 heavy chainconstant regions (SEQ ID NOs: 154 and 155).

The FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 divisions are shown in FIG.2A-3D.

Based on the sequence data, the germline genes from which each heavychain or light chain variable region was derived was determined. Theidentity of the germline genes are indicated next to the correspondinghybridoma line in FIGS. 2A-3D and each is represented by a unique SEQ IDNO. FIGS. 2A-3D also depict the determined amino acid sequences foradditional antibodies that were characterized.

Example 8 Determination of Isoelectric Points of Three Antibodies

The theoretical pIs of the antibodies based on amino acid sequence weredetermined to be 7.36 for 16F12; 8.47 for 21B12; and 6.84 for 31H4.

Example 9 Characterization of Binding of Antibodies to PCSK9

Having identified a number of antibodies that bind to PCSK9, severalapproaches were employed to quantify and further characterize the natureof the binding. In one aspect of the study, a Biacore affinity analysiswas performed. In another aspect of the study a KinExA® affinityanalysis was performed. The samples and buffers employed in thesestudies are presented in Table 6 below.

TABLE 6 [sample] [sample] sample mg/ml Buffer uM hPCSK9 1.26 PBS 16.6mPCSK9-8xHIS 1.44 PBS 18.9 cPCSK9-V5-6xHIS 0.22 PBS 2.9 16F12,anti-PCSK9 huIgG4 4.6 20 mM NaOAC, pH 31.9 5.2, 50 mM NaCl 21B12,anti-PCSK9 huIgG4 3.84 10 mM NAOAC, 27.0 pH 5.2, 9% Sucrose 31H4,anti-PCSK9 huIgG4 3.3 10 mM NAOAC, 22.9 pH 5.2, 9% Sucrose

BIAcore® Affinity Measurements

A BIAcore® (surface plasmon resonance device, Biacore, Inc., Piscataway,N.J.) affinity analysis of the 21B12 antibodies to PCSK9 described inthis Example was performed according to the manufacturer's instructions.

Briefly, the surface plasmon resonance experiments were performed usingBiacore 2000 optical biosensors (Biacore, GE Healthcare, Piscataway,N.J.). Each individual anti-PCSK9 antibody was immobilized to aresearch-grade CM5 biosensor chip by amine-coupling at levels that gavea maximum analyte binding response (Rmax) of no more than 200 resonanceunits (RU). The concentration of PCSK9 protein was varied at 2 foldintervals (the analyte) and was injected over the immobilized antibodysurface (at a flow rate of 100 μl/min for 1.5 minutes). Fresh HBS-Pbuffer (pH 7.4, 0.01 M Hepes, 0.15 M NaCl, 0.005% surfactant P-20,Biacore) supplemented with 0.01% BSA was used as binding buffer. Bindingaffinities of each anti-PCSK9 antibody were measured in separateexperiments against each of the human, mouse, and cynomolgus monkeyPCSK9 proteins at pH 7.4 (the concentrations used were 100, 50, 25,12.5, 6.25, 3.125, and 0 nM).

In addition, the binding affinities of antibody to human PCSK9 were alsomeasured at pH 6.0 with the pH 6.0 HBS-P buffer (pH 6.0, 0.01 M Hepes,0.15 M NaCl, 0.005% surfactant P-20, Biacore) supplemented with 0.01%BSA. The binding signal obtained was proportional to the free PCSK9 insolution. The dissociation equilibrium constant (K_(D)) was obtainedfrom nonlinear regression analysis of the competition curves using adual-curve one-site homogeneous binding model (KinExA® software,Sapidyne Instruments Inc., Boise, Id.) (n=1 for the 6.0 pH runs).Interestingly, the antibodies appeared to display a tighter bindingaffinity at the lower pH (where the Kd was 12.5, 7.3, and 29 pM for31H4, 21B12, and 16F12 respectively).

Antibody binding kinetic parameters including k_(a) (association rateconstant), k_(d) (dissociation rate constant), and K_(D) (dissociationequilibrium constant) were determined using the BIA evaluation 3.1computer program (BIAcore, Inc. Piscataway, N.J.). Lower dissociationequilibrium constants indicate greater affinity of the antibody forPCSK9. The K_(D) values determined by the BIAcore® affinity analysis arepresented in Table 7.1, shown below.

TABLE 7.1 Antibody hPCSK9 CynoPCSK9 mPCSK9 31H4 210 pM 190 pM 6 nM 21B12190 pM 360 pM 460 nM  16F12 470 pM 870 pM 6.4 nM  Table 7.2 depicts the k_(on) and k_(off) rates.

TABLE 7.2 — K_(on) (M-1 s-1) K_(off) (s-1) K_(D) 31H4.1, pH 7.4  2.45e+55.348e−5 210 pM 31H4.1, pH 6 5.536e+6 6.936e−5 12.5 pM  21B12.1, pH 7.43.4918e+4  6.634e−6 190 pM 21B12.1, pH 6 2.291e+6 1.676e−5  7.3 pM16F12.1, pH 7.4 1.064e+5 4.983e−5 470 pM 16F12.1, pH 6 2.392e+6 7.007e−5 29 pM

KinExA® Affinity Measurements

A KinExA® (Sapidyne Instruments, Inc., Boise, Id.) affinity analysis of16F12 and 31H4 was performed according to the manufacturer'sinstructions. Briefly, Reacti-Gel™ (6×) (Pierce) was pre-coated with oneof human, V5-tagged cyno or His-tagged mouse PCSK9 proteins and blockedwith BSA. 10 or 100 pM of either antibody 16F12 or antibody 31H4 and oneof the PCSK9 proteins was then incubated with various concentrations(0.1 pM-25 nM) of PCSK9 proteins at room temperature for 8 hours beforebeing passed through the PCSK9-coated beads. The amount of thebead-bound 16F12 or 31H4 was quantified by fluorescently (Cy5) labeledgoat anti-human IgG (H+ L) antibody (Jackson Immuno Research). Thebinding signal is proportional to the concentration of free 16F12 or31H4 at binding equilibrium. Equilibrium dissociation constant (K_(D))were obtained from nonlinear regression of the two sets of competitioncurves using a one-site homogeneous binding model. The KinExA® Prosoftware was employed in the analysis. Binding curves generated in thisanalysis are presented as FIGS. 4A-4F.

Both the 16F12 and 31H4 antibodies showed similar affinity to human andcyno PCSK9, but approximately 10-250 fold lower affinity to mouse PCSK9.Of the two antibodies tested using the KinExA® system, antibody 31H4showed higher affinity to both human and cyno PCSK9 with 3 and 2 pMK_(D), respectively. 16F12 showed slightly weaker affinity at 15 pMK_(D) to human PCSK9 and 16 pM K_(D) to cyno PCSK9.

The results of the KinExA® affinity analysis are summarized in Table8.1, shown below.

TABLE 8.1 hPCSK9 cPCSK mPCSK Sample K_(D) (pM) 95% CI K_(D) (pM) 95% CIK_(D) (pM) 95% CI 16F12 15 11~22 16 14~19 223 106~410 31H4.1 3 1~5 2 1~3500 400~620

In addition, a SDS PAGE was run to check the quality and quantity of thesamples and is shown in FIG. 5A. cPCSK9 showed around 50% less on thegel and also from the active binding concentration calculated fromKinExA® assay. Therefore, the K_(D) of the mAbs to cPCSK9 was adjustedas 50% of the active cPCSK9 in the present.

A BIAcore solution equilibrium binding assay was used to measure the Kdvalues for ABP 21B12. 21B12.1 showed little signal using KinExA assay,therefore, biacore solution equilibrium assay was applied. Since nosignificant binding was observed on binding of antibodies to immobilizedPCSK9 surface, 21B12 antibody was immobilized on the flow cell 4 of aCM5 chip using amine coupling with density around 7000 RU. Flow cell 3was used as a background control. 0.3, 1, and 3 nM of human PCSK9 orcyno PCSK9 were mixed with a serial dilutions of 21B12.1 antibodysamples (ranged from 0.001-25 nM) in PBS plus 0.1 mg/ml BSA, 0.005% P20.Binding of the free PCSK9 in the mixed solutions were measured byinjecting over the 21B12.1 antibody surface. 100% PCSK9 binding signalon 21B12.1 surface was determined in the absence of mAb in the solution.A decreased PCSK9 binding response with increasing concentrations of mAbindicated that PCSK9 binding to mAb in solution, which blocked PCSK9from binding to the immobilized peptibody surface. Plotting the PCSK9binding signal versus mAb concentrations, K_(D) was calculated fromthree sets of curves (0.3, 1 and 3 nM fixed PCSK9 concentration) using aone-site homogeneous binding model in KinExA Pro™ software. AlthoughcPCSK9 has lower protein concentration observed from KinExA assay andSDS-gel, its concentration was not adjusted here since the concentrationof cPCSK9 was not used for calculation of K_(D). The results aredisplayed in Table 8.2 below and in FIGS. 5B-5D. FIG. 5B depicts theresults from the solution equilibrium assay at three different hPCSK9concentrations for hPCSK9. FIG. 5C depicts a similar set of results formPCSK9. FIG. 5D depicts the results from the above biacore captureassay.

TABLE 8.2 hPCSK9 cPCSK mPCSK Sample K_(D) (pM) 95% CI K_(D) (pM) 95% CIK_(D) (pM) 95% CI 21B12.1 15 9~23 11 7~16 17000 —

Example 10 Epitope Binning

Competition ELISA was used for anti-PCSK9 antibody binning. Briefly, todetermine if two antibodies belong to the same epitope bin, one of theantibodies (mAb1) was first coated onto an ELISA plate (NUNC) at 2 μg/mlby overnight incubation. The plate was then washed and blocked with 3%BSA. Meanwhile, 30 ng/ml of biotinylated hPCSK9 was incubated with thesecond antibody (mAb2) for 2 hours at room temperature. The mixture wasapplied to coated mAb 1 and incubated for 1 hour at room temperature.The ELISA plate was then washed and incubated with Neutravidin-HRP(Pierce) at 1:5000 dilutions for 1 hour. After another wash, the platewas incubated with TMB substrate and signal was detected at 650 nm usinga Titertek plate reader. Antibodies with the same binding profiles weregrouped together into the same epitope bin. The results of the antibodybinning studies are presented in Table 8.3.

TABLE 8.3 Clone Bin 21B12.2 1 31H4 3 20D10 1 25A7.1 2 25A7.3 1 23G1 126H5 1 31D1 1 16F12 3 28D6 3 27A6 3 31G11 3 27B2 ND 28B12 3 22E2 31A12.2 1 3B6 1 3C4 4 9C9 1 9H6 1 13B5 6 13H1 7 17C2 1 19H9.2 1 23B5 125G4 1 26E10 1 27E7 1 27H5 1 30A4 1 30B9 1 31A4 5 31B12 5

Additional examination of the epitope binning was performed usingBIAcore. Three mAbs, 16F12, 21B12 and 31H4, were immobilized on flowcells 2, 3 and 4 with density around 8000 RU. 5 nM PCSK9 from human,mouse and cyno were injected over the mAb surfaces to reach around 100to 500 RU. 10 nM mAbs were then injected over the PCSK9 surface. Bindingof three mAbs to three different PCSK9 proteins over the three mAbs werethen recorded.

If the two mAbs had a similar epitope on the antigen, mAb 1 will notshow the binding to the antigen already bound to the mAb 2. If the twomAbs have the different epitope on the antigen, mAb 1 will show thebinding to the antigen bound to the mAb2. FIG. 5E depicts these epitopebinning results in graph form for three mAbs on human PCSk9. A similarpattern was observed for mPCSK9 and cPCSK9. As shown in the graph, 16F12and 31H4 appear to share a similar epitope, while 21B12 appears to havea different epitope.

Example 11 Efficacy of 31H4 and 21B12 for Blocking D374Y PCSK9/LDLRBinding

This example provides the IC50 values for two of the antibodies inblocking PCSK9 D374Y's ability to bind to LDLR. Clear 384 well plates(Costar) were coated with 2 micrograms/ml of goat anti-LDL receptorantibody (R&D Systems) diluted in buffer A (100 mM sodium cacodylate, pH7.4). Plates were washed thoroughly with buffer A and then blocked for 2hours with buffer B (1% milk in buffer A). After washing, plates wereincubated for 1.5 hours with 0.4 micrograms/ml of LDL receptor (R&DSystems) diluted in buffer C (buffer B supplemented with 10 mM CaCl2).Concurrent with this incubation, 20 ng/ml of biotinylated D374Y PCSK9was incubated with various concentrations of the 31H4 IgG2, 31H4 IgG4,21B12 IgG2 or 21B12 IgG4 antibody, which was diluted in buffer A, orbuffer A alone (control). The LDL receptor containing plates were washedand the biotinylated D374Y PCSK9/antibody mixture was transferred tothem and incubated for 1 hour at room temperature. Binding of thebiotinylated D374Y to the LDL receptor was detected by incubation withstreptavidin-HRP (Biosource) at 500 ng/ml in buffer C followed by TMBsubstrate (KPL). The signal was quenched with 1N HCl and the absorbanceread at 450 nm.

The results of this binding study are shown in FIGS. 6A-6D. Summarily,IC₅₀ values were determined for each antibody and found to be 199 pM for31H4 IgG2 (FIG. 6A), 156 pM for 31H4 IgG4 (FIG. 6B), 170 pM for 21B12IgG2 (FIG. 6C), and 169 pM for 21B12 IgG4 (FIG. 6D).

The antibodies also blocked the binding of wild-type PCSK9 to the LDLRin this assay.

Example 12 Cell LDL Uptake Assay

This example demonstrates the ability of various antigen bindingproteins to reduce LDL uptake by cells. Human HepG2 cells were seeded inblack, clear bottom 96-well plates (Costar) at a concentration of 5×10⁵cells per well in DMEM medium (Mediatech, Inc) supplemented with 10% FBSand incubated at 37° C. (5% CO2) overnight. To form the PCSK9 andantibody complex, 2 μg/ml of D374Y human PCSK9 was incubated withvarious concentrations of antibody diluted in uptake buffer (DMEM with1% FBS) or uptake buffer alone (control) for 1 hour at room temperature.After washing the cells with PBS, the D374Y PCSK9/antibody mixture wastransferred to the cells, followed by LDL-BODIPY (Invitrogen) diluted inuptake buffer at a final concentration of 6 μg/ml. After incubation for3 hours at 37° C. (5% CO2), cells were washed thoroughly with PBS andthe cell fluorescence signal was detected by Safire™ (TECAN) at 480-520nm (excitation) and 520-600 nm (emission).

The results of the cellular uptake assay are shown in FIGS. 7A-7D.Summarily, IC₅₀ values were determined for each antibody and found to be16.7 nM for 31H4 IgG2 (FIG. 7A), 13.3 nM for 31H4 IgG4 (FIG. 7B), 13.3nM for 21B12 IgG2 (FIG. 7C), and 18 nM for 21B12 IgG4 (FIG. 7D). Theseresults demonstrate that the applied antigen binding proteins can reducethe effect of PCSK9 (D374Y) to block LDL uptake by cells The antibodiesalso blocked the effect of wild-type PCSK9 in this assay.

Example 13 Serum Cholesterol Lowering Effect of the 31H4 Antibody in 6Day Study

In order to assess total serum cholesterol (TC) lowering in wild type(WT) mice via antibody therapy against PCSK9 protein, the followingprocedure was performed.

Male WT mice (C57BL/6 strain, aged 9-10 weeks, 17-27 g) obtained fromJackson Laboratory (Bar Harbor, Me.) were fed a normal chow(Harland-Teklad, Diet 2918) through out the duration of the experiment.Mice were administered either anti-PCSK9 antibody 31H4 (2 mg/ml in PBS)or control IgG (2 mg/ml in PBS) at a level of 10 mg/kg through themouse's tail vein at T=0. Naïve mice were also set aside as a naïvecontrol group. Dosing groups and time of sacrifice are shown in Table 9.

TABLE 9 Group Treatment Time point after dosing Number 1 IgG  8 hr 7 231H4  8 hr 7 3 IgG 24 hr 7 4 31H4 24 hr 7 5 IgG 72 hr 7 6 31H4 72 hr 7 7IgG 144 hr  7 8 31H4 144 hr  7 9 Naïve n/a 7

Mice were sacrificed with CO2 asphyxiation at the pre-determined timepoints shown in Table 9. Blood was collected via vena cava intoeppendorf tubes and was allowed to clot at room temperature for 30minutes. The samples were then spun down in a table top centrifuge at12,000×g for 10 minutes to separate the serum. Serum total cholesteroland HDL-C were measured using Hitachi 912 clinical analyzer andRoche/Hitachi TC and HDL-C kits.

The results of the experiment are shown in FIGS. 8A-8D. Summarily, miceto which antibody 31H4 was administered showed decreased serumcholesterol levels over the course of the experiment (FIG. 8A and FIG.8B). In addition, it is noted that the mice also showed decreased HDLlevels (FIG. 8C and FIG. 8D). For FIG. 8A and FIG. 8C, the percentagechange is in relation to the control IgG at the same time point(*P<0.01, # P<0.05). For FIG. 8B and FIG. 8D, the percentage change isin relation to total serum cholesterol and HDL levels measured in naïveanimals at t=0 hrs (*P<0.01, # P<0.05).

In respect to the lowered HDL levels, it is noted that one of skill inthe art will appreciate that the decrease in HDL in mice is notindicative that an HDL decrease will occur in humans and merely furtherreflects that the serum cholesterol level in the organism has decreased.It is noted that mice transport the majority of serum cholesterol inhigh density lipoprotein (HDL) particles which is different to humanswho carry most serum cholesterol on LDL particles. In mice themeasurement of total serum cholesterol most closely resembles the levelof serum HDL-C. Mouse HDL contains apolipoprotein E (apoE) which is aligand for the LDL receptor (LDLR) and allows it to be cleared by theLDLR. Thus, examining HDL is an appropriate indicator for the presentexample, in mice (with the understanding that a decrease in HDL is notexpected for humans). For example, human HDL, in contrast, does notcontain apoE and is not a ligand for the LDLR. As PCSK9 antibodiesincrease LDLR expression in mouse, the liver can clear more HDL andtherefore lowers serum HDL-C levels.

Example 14 Effect of Antibody 31H4 on LDLR Levels in a 6 Day Study

The present example demonstrates that an antigen binding protein altersthe level of LDLR in a subject, as predicted, over time. A Western blotanalysis was performed in order to ascertain the effect of antibody 31H4on LDLR levels. 50-100 mg of liver tissue obtained from the sacrificedmice described in Example 13 was homogenized in 0.3 ml of RIPA buffer(Santa Cruz Biotechnology Inc.) containing complete protease inhibitor(Roche). The homogenate was incubated on ice for 30 minutes andcentrifuged to pellet cellular debris. Protein concentration in thesupernatant was measured using BioRad protein assay reagents (BioRadlaboratories). 100 μg of protein was denatured at 70° C. for 10 minutesand separated on 4-12% Bis-Tris SDS gradient gel (Invitrogen). Proteinswere transferred to a 0.45 μm PVDF membrane (Invitrogen) and blocked inwashing buffer (50 mM Tris PH7.5, 150 mM NaCL, 2 mM CaCl₂ and 0.05%Tween 20) containing 5% non-fat milk for 1 hour at room temperature. Theblot was then probed with goat anti-mouse LDLR antibody (R&D system)1:2000 or anti-β actin (sigma) 1:2000 for 1 hour at room temperature.The blot was washed briefly and incubated with bovine anti-goat IgG-HRP(Santa Cruz Biotechnology Inc.) 1:2000 or goat anti-mouse IgG-HRP(Upstate) 1:2000. After a 1 hour incubation at room temperature, theblot was washed thoroughly and immunoreactive bands were detected usingECL plus kit (Amersham biosciences). The Western blot showed an increasein LDLR protein levels in the presence of antibody 31H4, as depicted inFIG. 9.

Example 15 Serum Cholesterol Lowering Effect of Antibody 31H4 in a 13Day Study

In order to assess total serum cholesterol (TC) lowering in wild type(WT) mice via antibody therapy against PCSK9 protein in a 13 day study,the following procedure was performed.

Male WT mice (C57BL/6 strain, aged 9-10 weeks, 17-27 g) obtained fromJackson Laboratory (Bar Harbor, Me.) were fed a normal chow(Harland-Teklad, Diet 2918) through out the duration of the experiment.Mice were administered either anti-PCSK9 antibody 31H4 (2 mg/ml in PBS)or control IgG (2 mg/ml in PBS) at a level of 10 mg/kg through themouse's tail vein at T=0. Naïve mice were also set aside as naïvecontrol group.

Dosing groups and time of sacrifice are shown in Table 10. Animals weresacrificed and livers were extracted and prepared as in Example 13.

TABLE 10 Group Treatment Time point after dosing Number Dose 1 IgG  72hr 6 10 mg/kg 2 31H4  72 hr 6 10 mg/kg 3 31H4  72 hr 6  1 mg/kg 4 IgG144 hr 6 10 mg/kg 5 31H4 144 hr 6 10 mg/kg 6 31H4 144 hr 6  1 mg/kg 7IgG 192 hr 6 10 mg/kg 8 31H4 192 hr 6 10 mg/kg 9 31H4 192 hr 6  1 mg/kg10 IgG 240 hr 6 10 mg/kg 11 31H4 240 hr 6 10 mg/kg 12 31H4 240 hr 6  1mg/kg 13 IgG 312 hr 6 10 mg/kg 14 31H4 312 hr 6 10 mg/kg 15 31H4 312 hr6  1 mg/kg 16 Naive n/a 6 n/a

When the 6 day experiment was extended to a 13 day study, the same serumcholesterol lowering effect observed in the 6 day study was alsoobserved in the 13 day study. More specifically, animals dosed at 10mg/kg demonstrated a 31% decrease in serum cholesterol on day 3, whichgradually returned to pre-dosing levels by day 13. FIG. 10A depicts theresults of this experiment. FIG. 10C depicts the results of repeatingthe above procedure with the mg/kg dose of 31H4, and with anotherantibody, 16F12, also at 10 mg/kg. Dosing groups and time of sacrificeare shown in Table 11.

TABLE 11 Group Treatment Time point after dosing Number Dose 1 IgG  24hr 6 10 mg/kg 2 16F12  24 hr 6 10 mg/kg 3 31H4  24 hr 6 10 mg/kg 4 IgG 72 hr 6 10 mg/kg 5 16F12  72 hr 6 10 mg/kg 6 31H4  72 hr 6 10 mg/kg 7IgG 144 hr 6 10 mg/kg 8 16F12 144 hr 6 10 mg/kg 9 31H4 144 hr 6 10 mg/kg10 IgG 192 hr 6 10 mg/kg 11 16F12 192 hr 6 10 mg/kg 12 31H4 192 hr 6 10mg/kg 13 IgG2 240 hr 6 10 mg/kg 14 16F12 240 hr 6 10 mg/kg 15 31H4 240hr 6 10 mg/kg 16 IgG2 312 hr 6 10 mg/kg 17 16F12 312 hr 6 10 mg/kg 1831H4 312 hr 6 10 mg/kg 19 Naive n/a 6 10 mg/kg

As shown in FIG. 10C both 16F12 and 31H4 resulted in significant andsubstantial decreases in total serum cholesterol after just a singledose and provided benefits for over a week (10 days or more). Theresults of the repeated 13 day study were consistent with the results ofthe first 13 day study, with a decrease in serum cholesterol levels of26% on day 3 being observed. For FIG. 10A and FIG. 10B, the percentagechange is in relation to the control IgG at the same time point(*P<0.01). For FIG. 10C, the percentage change is in relation to thecontrol IgG at the same time point (*P<0.05).

Example 16 Effect of Antibody 31H4 on HDL Levels in a 13 Day Study

The HDL levels for the animals in Example 15 were also examined. HDLlevels decreased in the mice. More specifically, animals dosed at 10mg/kg demonstrated a 33% decrease in HDL levels on day 3, whichgradually returned to pre-dosing levels by day 13. FIG. 10B depicts theresults of the experiment. There was a decrease in HDL levels of 34% onday 3. FIG. 10B depicts the results of the repeated 13 day experiment.

As will be appreciated by one of skill in the art, while the antibodieswill lower mouse HDL, this is not expected to occur in humans because ofthe differences in HDL in humans and other organisms (such as mice).Thus, the decrease in mouse HDL is not indicative of a decrease in humanHDL.

Example 17 Repeated Administration of Antibodies Produce ContinuedBenefits of Antigen Binding Peptides

In order to verify that the results obtained in the Examples above canbe prolonged for further benefits with additional doses, the Experimentsin Examples 15 and 16 were repeated with the dosing schedule depicted inFIG. 11A. The results are displayed in FIG. 11B. As can be seen in thegraph in FIG. 11B, while both sets of mice displayed a significantdecrease in total serum cholesterol because all of the mice received aninitial injection of the 31H4 antigen binding protein, the mice thatreceived additional injections of the 31H4 ABP displayed a continuedreduction in total serum cholesterol, while those mice that onlyreceived the control injection eventually displayed an increase in theirtotal serum cholesterol. For FIG. 11, the percentage change is inrelation to the naïve animals at t=0 hours (*P<0.01, **P<0.001).

The results from this example demonstrate that, unlike other cholesteroltreatment methods, in which repeated applications lead to a reduction inefficacy because of biological adjustments in the subject, the presentapproach does not seem to suffer from this issue over the time periodexamined. Moreover, this suggests that the return of total serumcholesterol or HDL cholesterol levels to baseline, observed in theprevious examples is not due to some resistance to the treatment beingdeveloped by the subject, but rather the depletion of the antibodyavailability in the subject.

Example 18 Epitope Mapping of Human Anti PCSK9 Antibodies

This example outlines methods for determining which residues in PCSK9are involved in forming or part of the epitope for the antigen bindingproteins disclosed herein to PCSK9.

In order to determine the epitopes to which certain of the ABPs of thepresent invention bind, the epitopes of the ABPs can be mapped usingsynthetic peptides derived from the specific PCSK9 peptide sequence.

A SPOTs peptide array (Sigma Genosys) can be used to study the molecularinteraction of the human anti-PCSK9 antibodies with their peptideepitope. SPOTs technology is based on the solid-phase synthesis ofpeptides in a format suitable for the systematic analysis of antibodyepitopes. Synthesis of custom arrayed oligopeptides is commericallyavailable from Sigma-Genosys. A peptide array of overlappingoligopeptides derived from the amino-acid sequence of the PCSK9 peptidecan be obtained. The array can comprise a series of 12-mer peptides asspots on a polypropylene membrane sheets. The peptide array can span theentire length of the PCSK9 mature sequence. Each consecutive peptide canbe offset by 1 residue from the previous one, yielding a nested,overlapping library of arrayed oligopeptides. The membrane carrying thepeptides can be reacted with different anti-PCSK9 antibodies (1micrograms/ml). The binding of the mAbs to the membrane-bound peptidescan be assessed by an enzyme-linked immunosorbent assay usingHRP-conjugated secondary antibody followed by enhanced chemiluminescence(ECL).

In addition, functional epitopes can be mapped by combinatorial alaninescanning. In this process, a combinatorial alanine-scanning strategy canbe used to identify amino acids in the PCSK9 protein that are necessaryfor interaction with anti-PCSK9 ABPs. To accomplish this, a second setof SPOTs arrays can be used for alanine scanning. A panel of variantpeptides with alanine substitutions in each of the 12 residues can bescanned as above. This will allow for the epitopes for the ABPs to thehuman PCSK9 to be mapped and identified.

In the alternative, given that it is possible that the epitope isconformational, a combination of alanine scanning and/or argininescanning, antibody FAB/PCSK9 co-crystallization, and limitedproteolysis/LC-MS (liquid chromatography mass spec.) can be employed toidentify the epitopes.

Example 19 Uses of PCSK9 Antibodies for the Treatment of CholesterolRelated Disorders

A human patient exhibiting a Cholesterol Related Disorder (in which areduction in cholesterol (such as serum cholesterol) can be beneficial)is administered a therapeutically effective amount of PCSK9 antibody,31H4 (or, for example, 21B12 or 16F12). At periodic times during thetreatment, the patient is monitored to determine whether the symptoms ofthe disorder has subsided. Following treatment, it is found thatpatients undergoing treatment with the PCSK9 antibody have reduced serumcholesterol levels, in comparison to patients that are not treated.

Example 20 Uses of PCSK9 Antibodies for the Treatment ofHypercholesterolemia

A human patient exhibiting symptoms of hypercholesterolemia isadministered a therapeutically effective amount of PCSK9 antibody, suchas 31H4 (or, for example, 21B12 or 16F12). At periodic times during thetreatment, the human patient is monitored to determine whether the serumcholesterol level has declined. Following treatment, it is found thatthe patient receiving the treatment with the PCSK9 antibodies hasreduced serum cholesterol levels in comparison to arthritis patients notreceiving the treatment.

Example 21 Uses of PCSK9 Antibodies for the Prevention of Coronary HeartDisease and/or Recurrent Cardiovascular Events

A human patient at risk of developing coronary heart disease isidentified. The patient is administered a therapeutically effectiveamount of PCSK9 antibody, such as 31H4 (or, for example, 21B12 or16F12), either alone, concurrently or sequentially with a statin, e.g.,simvastatin. At periodic times during the treatment, the human patientis monitored to determine whether the patient's total serum cholesterollevel changes. Throughout the preventative treatment, it is found thatthe patient receiving the treatment with the PCSK9 antibodies hasreduced serum cholesterol thereby reducing their risk to coronary heartdisases or recurrent cardiovascular events in comparison to patients notreceiving the treatment.

Example 22 Use of PCSK9 Antibodies as a Diagnostic Agent

An Enzyme-Linked Immunosorbent Assay (ELISA) for the detection of PCSK9antigen in a sample can used to diagnose patients exhibiting high levelsof PCSK9 production. In the assay, wells of a microtiter plate, such asa 96-well microtiter plate or a 384-well microtiter plate, are adsorbedfor several hours with a first fully human monoclonal antibody directedagainst PCSK9. The immobilized antibody serves as a capture antibody forany of the PCSK9 that may be present in a test sample. The wells arerinsed and treated with a blocking agent such as milk protein or albuminto prevent nonspecific adsorption of the analyte.

Subsequently the wells are treated with a test sample suspected ofcontaining the PCSK9, or with a solution containing a standard amount ofthe antigen. Such a sample may be, for example, a serum sample from asubject suspected of having levels of circulating antigen considered tobe diagnostic of a pathology.

After rinsing away the test sample or standard, the wells are treatedwith a second fully human monoclonal PCSK9 antibody that is labeled byconjugation with biotin. A monoclonal or mouse or other species origincan also be used. The labeled PCSK9 antibody serves as a detectingantibody. After rinsing away excess second antibody, the wells aretreated with avidin-conjugated horseradish peroxidase (HRP) and asuitable chromogenic substrate. The concentration of the antigen in thetest samples is determined by comparison with a standard curve developedfrom the standard samples.

This ELISA assay provides a highly specific and very sensitive assay forthe detection of the PCSK9 antigen in a test sample.

Determination of PCSK9 Protein Concentration in Subjects

A sandwich ELISA can quantify PCSK9 levels in human serum. Two fullyhuman monoclonal PCSK9 antibodies from the sandwich ELISA, recognizedifferent epitopes on the PCSK9 molecule. Alternatively, monoclonalantibodies of mouse or other species origin may be used. The ELISA isperformed as follows: 50 μL of capture PCSK9 antibody in coating buffer(0.1 M NaHCO₃, pH 9.6) at a concentration of 2 μg/mL is coated on ELISAplates (Fisher). After incubation at 4° C. overnight, the plates aretreated with 200 μL of blocking buffer (0.5% BSA, 0.1% Tween 20, 0.01%Thimerosal in PBS) for 1 hour at 25° C. The plates are washed (3×) using0.05% Tween 20 in PBS (washing buffer, WB). Normal or patient sera(Clinomics, Bioreclaimation) are diluted in blocking buffer containing50% human serum. The plates are incubated with serum samples overnightat 4° C., washed with WB, and then incubated with 100 μL/well ofbiotinylated detection PCSK9 antibody for 1 hour at 25° C. Afterwashing, the plates are incubated with HRP-Streptavidin for 15 minutes,washed as before, and then treated with 100 μL/well ofo-phenylenediamine in H₂O₂ (Sigma developing solution) for colorgeneration. The reaction is stopped with 50 μL/well of H₂SO₄ (2M) andanalyzed using an ELISA plate reader at 492 nm. Concentration of PCSK9antigen in serum samples is calculated by comparison to dilutions ofpurified PCSK9 antigen using a four parameter curve fitting program.

Determination of PCSK9 Variant Protein Concentration in Subjects

The steps outlined above can be performed using antibodies noted hereinthat bind to both the wild type PCSK9 and the variant PCSK9 (D374Y).Next, antibodies that bind to the wild type but not the mutant can beused (again using a similar protocol as outlined above) to determine ifthe PCSK9 present in the subject is wild type or the D374Y variant. Aswill be appreciatedy by one of skill in the art, results that arepositive for both rounds will be wild-type, while those that arepositive for the first round, but not the second round of antibodies,will include the D374Y mutation. There are high frequency mutations inthe population that are known and the could benefit particularly from anagent such as the ABPs disclosed herein.

Example 23 Use of PCSK9 Antigen Binding Protein for the Prevention ofHypercholesterolemia

A human patient exhibiting a risk of developing hypercholesterolemia isidentified via family history analysis and/or lifestyle, and/or currentcholesterol levels. The subject is regularly administered (e.g., onetime weekly) a therapeutically effective amount of PCSK9 antibody, 31H4(or, for example, 21B12 or 16F12). At periodic times during thetreatment, the patient is monitored to determine whether serumcholesterol levels have decreased. Following treatment, it is found thatsubjects undergoing preventative treatment with the PCSK9 antibody havelowered serum cholesterol levels, in comparison to subjects that are nottreated.

Example 24 PCSK9 ABPs Further Upregulated LDLR in the Presence ofStatins

This example demonstrates that ABPs to PCSK9 produced further increasesin LDLR availability when used in the presence of statins, demonstratingthat further benefits can be achieved by the combined use of the two.

HepG2 cells were seeded in DMEM with 10% fetal bovine serum (FBS) andgrown to ˜90% confluence. The cells were treated with indicated amountsof mevinolin (a statin, Sigma) and PCSK9 ABPs (FIGS. 12A-12C) in DMEMwith 3% FBS for 48 hours. Total cell lysates were prepared. 50 mg oftotal proteins were separated by gel electrophoresis and transferred toPVDF membrane. Immunoblots were performed using rabbit anti-human LDLreceptor antibody (Fitzgerald) or rabbit anti-human b-actin antibody.The enhanced chemiluminescent results are shown in the top panels ofFIGS. 12A-12C. The intensity of the bands were quantified by ImageJsoftware and normalized by b-actin. The relative levels of LDLR areshown in the lower panels of FIGS. 12A-12C. ABPs 21B12 and 31H4 arePCSK9 neutralizing antibodies, while 25A7.1 is a non-neutralizingantibody.

HepG2-PCSK9 cells were also created. These were stable HepG2 cell linetransfected with human PCSK9. The cells were seeded in DMEM with 10%fetal bovine serum (FBS) and grew to ˜90% confluence. The cells weretreated with indicated amounts of mevinolin (Sigma) and PCSK9 ABPs(FIGS. 12D-12F) in DMEM with 3% FBS for 48 hours. Total cell lysateswere prepared. 50 mg of total proteins were separated by gelelectrophoresis and transferred to PVDF membrane. Immunoblots wereperformed using rabbit anti-human LDL receptor antibody (Fitzgerald) orrabbit anti-human b-actin antibody. The enhanced chemiluminescentresults are shown in the top panels. The intensity of the bands werequantified by ImageJ software and normalized by b-actin.

As can be seen in the results depicted in FIGS. 12A-12F, increasingamounts of the neutralizing antibody and increasing amounts of thestatin generally resulted in increases in the level of LDLR. Thisincrease in effectiveness for increasing levels of the ABP is especiallyevident in FIGS. 12D-12F, in which the cells were also transfected withPCSK9, allowing the ABPs to demonstrate their effectiveness to a greaterextent.

Interestingly, as demonstrated by the results in the comparison of FIGS.12D-12F to 12A-12C, the influence of the ABP concentrations on LDLRlevels increased dramatically when PCSK9 was being produced by thecells. In addition, it is clear that the neutralizing ABPs (21B12 and31H4) resulted in a greater increase in LDLR levels, even in thepresence of statins, than the 25A7.1 ABP (a non-neutralizer),demonstrating that additional benefits can be achieved by the use ofboth statins and ABPs to PCSK9.

Example 25 Consensus Sequences

Consensus sequences were determined using standard phylogenic analysesof the CDRs corresponding to the V_(H) and V_(L) of anti-PCSK9 ABPs. Theconsensus sequences were determined by keeping the CDRs contiguouswithin the same sequence corresponding to a V_(H) or V_(L). Briefly,amino acid sequences corresponding to the entire variable domains ofeither V_(H) or V_(L) were converted to FASTA formatting for ease inprocessing comparative alignments and inferring phylogenies. Next,framework regions of these sequences were replaced with an artificiallinker sequence (“bbbbbbbbbb” placeholders, non-specific nucleic acidconstruct) so that examination of the CDRs alone could be performedwithout introducing any amino acid position weighting bias due tocoincident events (e.g., such as unrelated antibodies thatserendipitously share a common germline framework heritage) while stillkeeping CDRs contiguous within the same sequence corresponding to aV_(H) or V_(L). V_(H) or V_(L) sequences of this format were thensubjected to sequence similarity alignment interrogation using a programthat employs a standard ClutalW-like algorithm (see, Thompson et al.,1994, Nucleic Acids Res. 22:4673-4680). A gap creation penalty of 8.0was employed along with a gap extension penalty of 2.0. This programlikewise generated phylograms (phylogenic tree illustrations) based onsequence similarity alignments using either UPGMA (unweighted pair groupmethod using arithmetic averages) or Neighbor-Joining methods (see,Saitou and Nei, 1987, Molecular Biology and Evolution 4:406-425) toconstruct and illustrate similarity and distinction of sequence groupsvia branch length comparison and grouping. Both methods produced similarresults but UPGMA-derived trees were ultimately used as the methodemploys a simpler and more conservative set of assumptions.UPGMA-derived trees were generated where similar groups of sequenceswere defined as having fewer than 15 substitutions per 100 residues(see, legend in tree illustrations for scale) amongst individualsequences within the group and were used to define consensus sequencecollections. The results of the comparisons are depicted in FIGS.13A-13J. In FIG. 13E, the groups were chosen so that sequences in thelight chain that Glade are also a Glade in the heavy chain and havefewer than 15 substitutions.

As will be appreciated by one of skill in the art, the results presentedin FIGS. 13A-13J present a large amount of guidance as to the importanceof particular amino acids (for example, those amino acids that areconserved) and which amino acid positions can likely be altered (forexample, those positions that have different amino acids for differentABPs).

Example 26 Mouse Model for PCSK9 and ABP Ability to Lower LDL In Vivo

To generate mice which over-expressed human PCSK9, three week old WTC57B1/6 mice were injected via tail vein administration with variousconcentrations of adenoassociated virus (AAV), recombinantly modified toexpress human PCSK9, to determine the correct titer which would providea measurable increase of LDL-cholesterol in the mice. Using thisparticular virus that expressed human PCSK9, it was determined that4.5×10E12 pfu of virus would result in an LDL-cholesterol level ofapproximately 40 mg/dL in circulating blood (normal levels of LDL in aWT mice are approximately 10 mg/dL). The human PCSK9 levels in theseanimals was found to be approximately 13 ug/mL. A colony of mice weregenerated using this injection criteria.

One week after injection, mice were assessed for LDL-cholesterol levels,and randomized into different treatment groups. Animals were thenadministered, via tail vein injection, a single bolus injection ofeither 10 mg/kg or 30 mg/kg of 16F12, 21B12, or 31H4 antigen bindingproteins. IgG2 ABP was administered in a separate group of animals as adosing control. Subgroups of animals (n=6-7) were then euthanized at 24and 48 hours after ABP administration. There were no effects onLDL-cholesterol levels following IgG2 administration at either dose.Both 31H4 and 21B12 demonstrated significant LDL-cholesterol lowering upto and including 48 hours post-administration, as compared to IgG2control (shown in FIGS. 14A and 14B at two different doses). 16F12 showsan intermediary LDL-cholesterol lowering response, with levels returningto baseline of approximately 40 mg/dL by the 48 hour time point. Thisdata is consistent with in vitro binding data (Biacore and Kinexa),which shows near equivalent binding affinity between 31H4 and 21B12, anda lesser affinity of 16F12 to human PCSK9.

As can be seen in the results, total cholesterol and HDL-cholesterolwere reduced by the PCSK9 ABPs in the model (both total and HDL-C areelevated above WT mice due to the overexpression of PCSK9). Whilecholesterol lowering in this model appears to occur over a relativelyshort period of time, this is believed to be due to the levels of humanPCSK9 that are present, which are supraphysiologically high in thismodel. In addition, given that the expression is governed by AAV, thereis no regulation of PCSK9 expression. In these figures, (*) denotes aP<0.05, and (**) denotes a P<0.005 as compared to LDL-cholesterol levelsobserved in IgG2 control injected animals at the same time point. The 13microgram/ml level of serum human PCSK9 in the mice corresponds to anapproximately 520-fold increase above the endogenous mouse PCSK9 levels(˜25 ng/ml), and an approximately 75-fold increase above average humanserum levels (˜175 ng/ml). Thus, the antigen binding proteins should beeven more effective in humans.

As will be appreciated by one of skill in the art, the above resultsdemonstrate that appropriateness of the mouse model for testing theantigen binding protein's ability to alter serum cholesterol in asubject. One of skill in the art will also recognize that the use ofmouse HDL to monitor serum cholesterol levels in a mouse, while usefulfor monitoring mouse serum cholesterol levels, is not indicative of theABPs impact on human HDL in humans. For example, Cohen et al. (“Sequencevariations in PCSK9, low LDL, and protection against coronary heartdisease”, N Engl J Med, 354:1264-1272, 2006) demonstrated the lack ofany effect of the PCSK9 loss-of-function mutations on human HDL levels(the entirety of which is incorporated by reference). Thus, one of skillin the art will appreciate that the ability of the ABP to lower mouseHDL (which lack LDL) is not indicative of the ABP's ability to lowerhuman HDL. Indeed, as shown by Cohen, this is unlikely to occur forneutralizing antibodies in humans.

Example 27 31H4 and 21B12 Bind to the ProCat Region of PCSK9

The present example describes one method for determining where variousantibodies bind to PCSK9.

The ProCat (31-449 of SEQ ID NO: 3) or V domain (450-692 of SEQ ID NO:3) of the PCSK9 protein was combined with either antibody 31H4 or 21B12.The samples were analyzed by Native PAGE for complex formation. As canbe seen in FIG. 16A and FIG. 16B, gel shifts were present for theProCat/31H4 and ProCat/21B12 samples, demonstrating that the antibodiesbound to the ProCat domain.

Example 28 The LDLR EGFa Domain Binds to the Catalytic Domain of PCSK9

The present example presents the solved crystal structure of PCSK9ProCat (31-454 of SEQ ID NO: 3) bound to the LDLR EGFa domain (293-334)at 2.9 Å resolution (the conditions for which are described in the belowExamples).

A representation of the structure of PCSK9 bound to EGFa is shown inFIG. 17. The crystal structure (and its depiction in FIG. 17) revealsthat the EGFa domain of LDLR binds to the catalytic domain of PCSK9. Inaddition, the interaction of PCSK9 and EGFa appears to occur across asurface of PCSK9 that is between residues D374 and S153 in the structuredepicted in FIG. 17.

Specific core PCSK9 amino acid residues of the interaction interfacewith the LDLR EGFa domain were defined as PCSK9 residues that are within5 Å of the EGFa domain. The core residues are as follows: S153, I154,P155, R194, D238, A239, I369, S372, D374, C375, T377, C378, F379, V380,and S381.

Boundary PCSK9 amino acid residues of the interaction interface with theLDLR EGFa domain were defined as PCSK9 residues that are 5-8 Å from theEGFa domain. The boundary residues are as follows: W156, N157, L158,E159, H193, E195, H229, R237, G240, K243, D367, I368, G370, A371, S373,S376, and Q382. Residues that are underlined are nearly or completelyburied within PCSK9.

As will be appreciated by one of skill in the art, the results from thisexample demonstrate where PCSK9 and EGFa interact. Thus, antibodies thatinteract with or block any of these residues can be useful as antibodiesthat inhibit the interaction between PCSK9 and the EGFa domain of LDLR(and/or LDLR generally). In some embodiments, antibodies that, whenbound to PCSK9, interact with or block any of the above residues or arewithin 15-8, 8, 8-5, or 5 angstroms of the above residues arecontemplated to provide useful inhibition of PCSK9 binding to LDLR.

Example 29 31H4 Interacts with Amino Acid Residues from Both the Pro-and Catalytic Domains of PCSK9

The present example presents the crystal structure of full length PCSK9(N533A mutant of SEQ ID NO: 3) bound to the Fab fragment of 31H4,determined to 2.3 Å resolution (the conditions for which are describedin the below Examples). This structure, depicted in FIGS. 18A and 18B,shows that 31H4 binds to PCSK9 in the region of the catalytic site andmakes contacts with amino acid residues from both the prodomain andcatalytic domain.

The depicted structure also allows one to identify specific core PCSK9amino acid residues for the interaction interface of 31H4 with PCSK9.This was defined as residues that are within 5 Å of the 31H4 protein.The core residues are as follows: W72, F150, A151, Q152, T214, R215,F216, H217, A220, S221, K222, S225, H226, C255, Q256, G257, K258, N317,F318, T347, L348, G349, T350, L351, E366, D367, D374, V380, S381, Q382,S383, and G384.

The structures were also used to identify boundary PCSK9 amino acidresidues for the interaction interface with 31H4. These residues werePCSK9 residues that were 5-8 Å from the 31H4 protein. The boundaryresidues are as follows: K69, D70, P71, S148, V149, D186, T187, E211,D212, G213, R218, Q219, C223, D224, G227, H229, L253, N254, G259, P288,A290, G291, G316, R319, Y325, V346, G352, T353, G365, I368, I369, S372,S373, C378, F379, T385, S386, and Q387. Amino acid residues completelyburied within the PCSK9 protein are underlined.

As will be appreciated by one of skill in the art, FIG. 18B depicts theinteraction between the CDRs on the antigen binding protein and PCSK9.As such, the model allows one of skill in the art to identify theresidues and/or CDRs that are especially important in the paratope, andwhich residues are less critical to the paratope. As can be seen in FIG.18B, the heavy chain CDR1, CDR2, and CDR3 are most directly involved inthe antigen binding protein's binding to the epitope, with the CDRs fromthe light chain being relatively far away from the epitope. As such, itis probable that larger variations in the light chain CDRs are possible,without unduly interfering with the binding of the antigen bindingprotein to PCSK9. In some embodiments, residues in the structures thatdirectly interact are conserved (or alternatively conservativelyreplaced) while residues that are not directly interacting with oneanother can be altered to a greater extent. As such, one of skill in theart, given the present teachings, can predict which residues and areasof the antigen binding proteins can be varied without unduly interferingwith the antigen binding protein's ability to bind to PCSK9. Forexample, those residues that are located closest to PCSK9 when theantigen binding protein is bound to PCSK9 are those that likely play amore important role in the binding of the antigen binding protein toPCSK9. As above, these residues can be divided into those that arewithin 5 angstroms of PCSK9 and those that are between 5 and 8angstroms. Specific core 31H4 amino acid residues of the interactioninterface with PCSK9 were defined as 31H4 residues that are within 5 Åof the PCSK9 protein. For the heavy chain, the residues that are within5 angstroms include the following: T28, S30, S31, Y32, S54, S55, S56,Y57, I58, S59, Y60, N74, A75, R98, Y100, F102, W103, S104, A105, Y106,Y107, D108, A109, and D111. For the light chain, those residues that arewithin 5 angstroms include the following: L48, S51, Y93, and S98. Forthe heavy chain, those residues that are 5-8 Å from the PCSK9 proteininclude the following: G26, F27, F29, W47, S50, I51, S52, S53, K65, F68,T69, I70, S71, R72, D73, K76, N77, D99, D101, F110, and V112. For thelight chain, those residues that are within 5-8 angstroms of PCSK9include A31, G32, Y33, D34, H36, Y38, I50, G52, N55, R56, P57, S58, D94,S95, S96, L97, G99, and S100.

As will be appreciated by one of skill in the art, the results fromExample 29 demonstrate where antibodies to PCSK9 can interact on PCSK9and still block PCSK9 from interacting with EGFa (and thus LDLR). Thus,antigen binding proteins that interact with any of these PCSK9 residues,or that block any of these residues (e.g., from other antigen bindingproteins that bind to these residues), can be useful as antibodies thatinhibit the interaction of PCSK9 and EGFa (and LDLR accordingly). Thus,in some embodiments, antigen binding proteins that interact with any ofthe above residues or interact with residues that are within 5 Å of theabove residues are contemplated to provide useful inhibition PCSK9binding to LDLR. Similarly, antigen binding proteins that block any ofthe above residues (which can be determined, for example, via acompetition assay) can also be useful for inhibition of the PCSK9/LDLRinteraction.

Example 30 21B12 Binds to the Catalytic Domain of PCSK9, has a DistinctBinding Site from 31H4 and can Bind to PCSK9 Simultaneously with 31H4

The present example presents the crystal structure of PCSK9 ProCat(31-449 of SEQ ID NO: 3) bound to the Fab fragments of 31H4 and 21B12,determined at 2.8 Å resolution (the conditions for which are describedin the below Examples). This crystal structure, depicted in FIG. 19A andFIG. 19B, shows that 31H4 and 21B12 have distinct binding sites on PCSK9and that both antigen binding proteins can bind to PCSK9 simultaneously.The structure shows that 21B12 interacts with amino acid residues fromPCSK9's catalytic domain. In this structure, the interaction betweenPCSK9 and 31H4 is similar to what was observed above.

Specific core PCSK9 amino acid residues of the interaction interfacewith 21B12 were defined as PCSK9 residues that are within 5 Å of the21B12 protein. The core residues are as follows: S153, S188, I189, Q190,S191, D192, R194, E197, G198, R199, V200, D224, R237, D238, K243, S373,D374, S376, T377, and F379.

Boundary PCSK9 amino acid residues of the interaction interface with21B12 were defined as PCSK9 residues that were 5-8 Å from the 21B12protein. The boundary residues are as follows: I154, T187, H193, E195,I196, M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372,C375, and C378. Amino acid residues nearly or completely buried withinthe PCSK9 protein are underlined.

As will be appreciated by one of skill in the art, FIG. 19B depicts theinteraction between the CDRs on the antigen binding protein and PCSK9.As such, the model allows one of skill in the art to identify theresidues and/or CDRs which are especially important for the paratope andwhich residues are less critical to the paratope. As can be seen in thestructure, heavy chain CDR2 and light chain CDR1 appear to closelyinteract with the epitope. Next, heavy chain CDR1, heavy chain CDR3 andlight chain CDR3, appear to be close to the epitope, but not as close asthe first set of CDRs. Finally, light chain CDR2 appears to be somedistance from the epitope. As such, it is probable that largervariations in the more distant CDRs are possible without undulyinterfering with the binding of the antigen binding protein to PCSK9. Insome embodiments, residues in the structures that directly interact areconserved (or alternatively conservatively replaced) while residues thatare not directly interacting with one another can be altered to agreater extent. As such, one of skill in the art, given the presentteachings, can predict which residues and areas of the antigen bindingproteins can be varied without unduly interfering with the antigenbinding protein's ability to bind to PCSK9. For example, those residuesthat are located closest to PCSK9 when the antigen binding protein isbound to PCSK9 are those that likely play a more important role in thebinding of the antigen binding protein to PCSK9. As above, theseresidues can be divided into those that are within 5 angstroms of PCSK9and those that are between 5 and 8 angstroms. Specific core 21B12 aminoacid residues of the interaction interface with PCSK9 were defined as21B12 residues that are within 5 Å of the PCSK9 protein. For the heavychain, the residues that are within 5 angstroms include the following:T30, S31, Y32, G33, W50, S52, F53, Y54, N55, N57, N59, R98, G99, Y100,and G101. For the light chain, those residues that are within 5angstroms include the following: G30, G31, Y32, N33, S34, E52, Y93, T94,S95, T96, and S97. For the heavy chain, those residues that are 5-8 Åfrom the PCSK9 protein include the following: T28, L29, I34, S35, W47,V51, G56, T58, Y60, T72, M102, and D103. For the light chain, thoseresidues that are within 5-8 angstroms of PCSK9 include the following:S26, V29, V35, Y51, N55, S92, M98, and V99.

As will be appreciated by one of skill in the art, the results fromExample 30 demonstrate where antigen binding proteins to PCSK9 caninteract on PCSK9 and still block PCSK9 from interacting with EGFa (andthus LDLR). Thus, antigen binding proteins that interact with any ofthese PCSK9 residues or that block any of these residues can be usefulas antibodies that inhibit the interaction of PCSK9 and EGFa (and LDLRaccordingly). Thus, in some embodiments, antibodies that interact withany of the above residues or interact with residues that are within 5 Åof the above residues are contemplated to provide useful inhibitionPCSK9 binding to LDLR. Similarly, antigen binding proteins that blockany of the above residues (which can be determined, for example, via acompetition assay) can also be useful for inhibition of PCSK9/LDLRinteraction.

Example 31 Interaction Between EGFa, PCSK9, and the Antibodies

The structure of the ternary complex (PCSK9/31H4/21B12) from the aboveexample was overlaid on the PCSK9/EGFa structure (determined asdescribed in Example 28) and the result of this combination is depictedin FIG. 20A. This figure demonstrates areas on PCSK9 which can beusefully targeted to inhibit PCSK9 interaction with EGFa. The figureshows that both 31H4 and 21B12 partially overlap with the position ofthe EGFa domain of LDLR and sterically interfere with its binding toPCSK9. In addition, as can be seen in the structures, 21B12 directlyinteracts with a subset of amino acid residues that are specificallyinvolved in binding to the LDLR EGFa domain.

As noted above, analysis of the crystal structures identified specificamino acids involved in the interaction between PCSK9 and the partnerproteins (the core and boundary regions of the interface on the PCSK9surface) and the spatial requirements of these partner proteins tointeract with PCSK9. The structures suggest ways to inhibit theinteraction between PCSK9 and the LDLR. First, as noted above, bindingan agent to PCSK9 where it shares residues in common with the bindingsite of the EGFa domain of the LDLR would inhibit the interactionbetween PCSK9 and the LDLR. Second, an agent that binds outside of theresidues in common can sterically interfere with the EGFa domain orregions of the LDLR that are either N- or C-terminal to the EGFa domainto prevent the interaction between PCSK9 and the LDLR.

In some embodiments, the residues that are involved in both EGFa bindingand are close to the areas where the above noted antigen bindingproteins bind are especially useful for manipulating PCSK9 binding toLDLR. For example, amino acid residues from interfaces in common in boththe core region and boundary region for the different binding partnersare listed in Table 12 below. Amino acid residues completely buriedwithin the PCSK9 protein are underlined.

TABLE 12 Parameters Amino acid position(s) 31H4/EGFa both under 5 ÅD374, V380, S381 31H4 under 5 Å/EGFa 5-8 Å D367, Q382 31H4 at 5-8 Å/EGFaunder 5 Å I369, S372, C378, F379 31H4/EGFa both at 5-8 Å H229, S37321B12/EGFa both under 5 Å S153, R194, D238, D374, T377, F379 21B12 under5 Å/EGFa 5-8 Å R237, K243, S373, S376 21B12 at 5-8 Å/EGFa under 5 ÅI154, A239, I369, S372, C375, C378 21B12/EGFa both at 5-8 Å H193, E195

As will be appreciated by one of skill in the art, in some embodiments,the antigen binding proteins bind to and/or block at least one of theabove noted residues.

Example 32 Structural Interaction of LDLR and PCSK9

A model of full length PCSK9 bound to a full length representation ofthe LDLR was made using the PCSK9 ProCat (31-454 of SEQ ID NO: 3)/EGFacomplex structure. The structure of full length PCSK9¹ (Piper, D. E. etal. The crystal structure of PCSK9: a regulator of plasmaLDL-cholesterol. Structure 15, 545-52 (2007)) was overlaid onto thePCSK9 ProCat 31-454 from the complex and the structure of the LDLR inits low pH conformation (Rudenko, G. et al. Structure of the LDLreceptor extracellular domain at endosomal pH. Science 298, 2353-8(2002)) was overlaid onto the EGFa domain from the complex. Depictionsof the model are shown in FIGS. 20B and 20C. The EGFa domain isindicated by the box in the figure. The figures show regions of the LDLRoutside of the immediate EGFa binding domain that lie in close proximityto PCSK9. FIGS. 20D-20F show the above interaction, along with meshsurface representations of antibody 31H4 and 21B12 from three differentangles. As is clear from the depictions, not only can the antibodyinteract and/or interfere with LDLR's interaction with PCSK9 at theactual binding site, but other steric interactions appear to occur aswell.

In light of the above results, it is clear that antigen binding proteinsthat bind to PCSK9 can also inhibit the interaction between PCSK9 andthe LDLR by clashing with various regions of the LDLR (not just the siteat which LDLR and PCSK9 interact). For example, it can clash with repeat7 (R7), the EGFb domain, and/or the β-propeller domain.

Embodiments of Antigen Binding Molecules that Bind to or Block EGFaInteraction with PCSK9

As will be appreciated by one of skill in the art, Examples 28-32, andtheir accompanying figures, provide a detailed description of how andwhere EGFa interacts with PCSK9 and how two representative neutralizingantigen binding proteins, 21B12 and 31H4 interact with PCSK9 and producetheir neutralizing effect. As such, one of skill in the art will readilybe able to identify antigen binding molecules that can similarly reducethe binding between EGFa (including LDLR) and PCSK9 by identifying otherantigen binding molecules that bind at or near at least one of the samelocations on PCSK9. While the relevant locations (or epitopes) on PCSK9are identified in the figures and the present description, it can alsobe advantageous to describe these sites as being within a set distancefrom residues that have been identified as close to the EGFa bindingsite. In some embodiments, an antigen binding molecule will bind to orwithin 30 angstroms of one or more of the following residues (numberingin reference to SEQ ID NO: 3): S153, I154, P155, R194, D238, A239, I369,S372, D374, C375, T377, C378, F379, V380, S381, W156, N157, L158, E159,H193, E195, H229, R237, G240, K243, D367, I368, G370, A371, S373, S376,Q382, W72, F150, A151, Q152, T214, R215, F216, H217, A220, S221, K222,S225, H226, C255, Q256, G257, K258, N317, F318, T347, L348, G349, T350,L351, E366, D367, D374, V380, S381, Q382, S383, G384, K69, D70, P71,S148, V149, D186, T187, E211, D212, G213, R218, Q219, C223, D224, G227,H229, L253, N254, G259, P288, A290, G291, G316, R319, Y325, V346, G352,T353, G365, I368, I369, S372, S373, C378, F379, T385, S386, Q387, S153,S188, I189, Q190, S191, D192, R194, E197, G198, R199, V200, D224, R237,D238, K243, S373, D374, S376, T377, F379, I154, T187, H193, E195, I196,M201, V202, C223, T228, S235, G236, A239, G244, M247, I369, S372, C375,or C378. In some embodiments, the antigen binding molecule binds within30 angstroms of one or more of the following residues (numbering inreference to SEQ ID NO: 3): S153, I154, P155, R194, D238, A239, I369,S372, D374, C375, T377, C378, F379, V380, S381, W156, N157, L158, E159,H193, E195, H229, R237, G240, K243, D367, I368, G370, A371, S373, S376,or Q382. In some embodiments, the antigen binding molecule binds within30 angstroms of one or more of the following residues (numbering inreference to SEQ ID NO: 3): W72, F150, A151, Q152, T214, R215, F216,H217, A220, S221, K222, S225, H226, C255, Q256, G257, K258, N317, F318,T347, L348, G349, T350, L351, E366, D367, D374, V380, S381, Q382, S383,G384, K69, D70, P71, S148, V149, D186, T187, E211, D212, G213, R218,Q219, C223, D224, G227, H229, L253, N254, G259, P288, A290, G291, G316,R319, Y325, V346, G352, T353, G365, I368, I369, S372, S373, C378, F379,T385, S386, or Q387. In some embodiments, the antigen binding moleculebinds within 30 angstroms of one or more of the following residues(numbering in reference to SEQ ID NO: 3): S153, S188, I189, Q190, S191,D192, R194, E197, G198, R199, V200, D224, R237, D238, K243, S373, D374,S376, T377, F379, I154, T187, H193, E195, I196, M201, V202, C223, T228,S235, G236, A239, G244, M247, I369, S372, C375, or C378.

In some embodiments, the antigen binding molecule binds within 30,30-25, 25-20, 20-15, 15-8,8,8-5,5,5-4, 4 or less angstroms from one ormore of the above residues. In some embodiments, the antigen bindingmolecule, when bound to PCSK9, is within at least one of the abovedistances, for more than one of the above noted residues. For example,in some embodiments, the antigen binding molecule is within one of therecited distances (e.g., 30, 30-25, 25-20, 20-15, 15-8,8,8-5,5,5-4, 4 orless) for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60,60-65, 65-70, 70-75 or more of the above residues. In some embodiments,the antigen binding molecule is within one of the recited distances forat least 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90,90-95, 95-99, 99-100% of the residues identified in each group ofsubgroup thereof (such as only those surface residues in the group).Unless specifically stated otherwise, the distance between the antigenbinding molecule and PCSK9 is the shortest distance between thecovalently bonded atom on PCSK9 and the covalently bonded atom of theantigen binding molecule that are the closest atoms of PCSK9 and theantigen binding molecule. Similarly, unless specifically statedotherwise, the distance between a residue (on the antigen bindingmolecule or PCSK9) and another protein (either PCSK9 or the antigenbinding molecule respectively), is the distance from the closest pointon the identified residue to the closest covalently bonded part of theother protein. In some embodiments, the distance can be measured fromthe backbone of the amino acid chains. In some embodiments, the distancecan be measured between an edge of the paratope and an edge (closest toone another) of the epitope. In some embodiments, the distance can bemeasured between the center of the surface of the paratope and thecenter of the surface of the epitope. As will be appreciated by one ofskill in the art, the present description is applicable for each of theindividual sets of residues listed herein. For example, the above rangesare contemplated generally and specifically for the 8 angstrom residueslisted in Examples 28-32 and the 5 angstrom residues listed in Examples28-32.

In some embodiments, the antigen binding molecule binds to a surface onPCSK9 that is bound by at least one of EGFa, 21B12, or 31H4. In someembodiments, the antigen binding molecule binds to PCSK9 at a locationthat overlaps with the interaction locations between PCSK9 and EFGa, Ab31H4, and/or Ab 21B12 (as described in the above examples and figures).In some embodiments, the antigen binding molecule binds to PCSK9 at aposition that is further away from one of the above recited residues. Insome embodiments, such an antigen binding molecule can still be aneffective neutralizing antigen binding molecule.

In some embodiments, the structure of the catalytic domain of PCSK9 canbe described as generally being triangular (as shown in FIG. 19A). Thefirst side of the triangle is shown as being bound by 31H4. The secondside of the triangle is shown as being bound by 21B12, and the thirdside of the triangle is positioned toward the bottom of the page,immediately above the “FIG. 19A” label. In some embodiments, antigenbinding molecules that bind to the first and/or second sides of thecatalytic domain of PCSK9 can be useful as neutralizing antibodies asthey can either directly or sterically interfere with EGFa's binding toPCSK9. As will be appreciated by one of skill in the art, when theantigen binding molecules are large enough, such as a full antibody, theantigen binding molecule need not directly bind to the EGFa binding sitein order to interfere with the binding of EGFa to PCSK9.

As will be appreciated by one of skill in the art, while the EGFa domainof the LDLR has been used in many of the examples, the models andstructures are still applicable to how the full length LDLR protein willinteract with PCSK9. Indeed, the additional structure present on thefull length LDLR protein presents additional protein space that canfurther be blocked by one of the antigen binding molecules. As such, ifthe antigen binding molecule blocks or inhibits binding of EGFa toPCSK9, it will likely be at least as, if not more, effective with thefull length LDLR protein. Similarly, antigen binding molecules that arewithin a set distance or block various residues that are relevant forinhibiting EGFa binding, will likely be as effective, if not moreeffective, for the full length LDLR.

As will be appreciated by one of skill in the art, any molecule thatblocks or binds to the above noted PCSK9 residues (or within the reciteddistances), or that inhibits one or more of the interactions noted inthe above examples and figures, can be used to inhibit the interactionof EGFa (or LDLR generally) and PCSK9. As such, the molecule need not belimited to an antigen binding “protein,” as any antigen binding moleculecan also serve the required purpose. Examples of antigen bindingmolecules include aptamers, which can be either oligonucleic acid orpeptide molecules. Other examples of antigen binding molecules includeavimers, peptibodies, small molecules and polymers, and modifiedversions of EGFa that can increase its affinity to PCSK9 and/orhalf-life, such as mutation of amino acids, glycosylation, pegylation,Fc fusions, and avimer fusions. As will be appreciated by one of skillin the art, in some embodiments LDLR is not an antigen binding molecule.In some embodiments, binding subsections of LDLR are not antigen bindingmolecules, e.g., EGFa. In some embodiments, other molecules throughwhich PCSK9 signals in vivo are not antigen binding molecules. Suchembodiments will be explicitly identified as such.

Example 33 Expression and Purification of Protein Samples

The present example describes some embodiments for how the variousembodiments of the PCSK9 proteins/variants were made and purified(including the LDLR EGFa domain). PCSK9 proteins/variants (e.g., PSCK931-692 N533A, PCSK9 449TEV and PCSK9 ProCat 31-454) were expressed inbaculovirus infected Hi-5 insect cells with an N-terminal honeybeemelittin signal peptide followed by a His₆ tag. The PCSK9 proteins werepurified by nickel affinity chromatography, ion exchange chromatographyand size exclusion chromatography. The melittin-His₆ tag was removedduring purification by cleavage with TEV protease. The construct PCSK9449TEV was used to generate PCSK9 ProCat (31-449) and V domain (450-692)samples. This construct had a TEV protease cleavage site insertedbetween PCSK9 residues 449 and 450. For the full length N555A variantfor crystallography, the PCSK9 31-454 fragment, and the PCSK9 449TEVvariant for crystalography, the post rTEV protein product also includedan initial GAMG sequence. Thus, post rTEV cleavage, these proteins wereGAMG-PCSK9. Furthermore, the PCSK9 449TEV protein included the sequence“ENLYFQ” (SEQ ID NO: 403) inserted between positions H449 and G450 ofSEQ ID NO: 3. After cleavage with rTEV, the PCSK9 ProCat proteingenerated from this construct was GAMG-PCSK9 (31-449)-ENLYFQ and the Vdomain generated from this construct was PCSK9 (450-692) of SEQ ID NO:3.

The 21B12 and 31H4Fab fragments were expressed in E. coli. Theseproteins were purified by nickel affinity chromatography, size exclusionchromatography and ion exchange chromatography.

The LDLR EGFa domain (293-334) was expressed as a GST fusion protein inE. coli. The EGFa domain was purified by ion exchange chromatography,glutathione sepharose affinity chromatography and size exclusionchromatography. The GST protein was removed during the purification bycleavage with PreScission protease.

Example 34 Complex Formation and Crystallization

The present example describes how complexes and crystals used in theabove structure examination Examples were made.

The PCSK9 31-692 N533A/31H4 complex was made by mixing a 1.5 molarexcess of the 31H4Fab with PCSK9. The complex was purified by sizeexclusion chromatography to remove excess 31H4Fab. The PCSK9 31-692N533A/31H4 complex crystallizes in 0.1 M Tris pH 8.3, 0.2 M sodiumacetate, 15% PEG 4000, 6% dextran sulfate sodium salt (Mr 5000).

The PCSK9 ProCat 31-449/31H4/21B12 complex was made by first mixing a1.5 molar excess of 31H4Fab with PCSK9 31-449. The complex was separatedfrom excess 31H4 by purification on a size exclusion chromatographycolumn. A 1.5 molar excess of 21B12 Fab was then added to the PCSK931-449/31H4 complex. The ternary complex was separated from excess 21B12by purification on a size exclusion chromatography column. The PCSK9ProCat 31-449/31H4/21B12 complex crystallizes in 0.1 M Tris pH 8.5, 0.2M ammonium phosphate monobasic, 50% MPD.

The PCSK9 ProCat 31-454/EGFa complex was made by mixing a 1.2 molarexcess of EGFa domain with PCSK9 31-454. The PCSK9 ProCat 31-454/EGFadomain complex crystallizes in 0.2 M potassium formate, 20% PEG 3350.

Example 35 Data Collection and Structure Determination

The present example describes how the datasets were collected and thestructures determined for the above structure examination Examples.

Initial datasets for the PCSK9 31-692 N533A/31H4 and PCSK9 ProCat31-449/31H4/21B12 crystals were collected on a Rigaku FR-E X-ray source.The PCSK9 ProCat 31-454/EGFa dataset and higher resolution datasets forthe PCSK9 31-692 N533A/31H4 and PCSK9 ProCat 31-449/31H4/21B12 crystalswere collected at the Berkeley Advanced Light Source beamline 5.0.2. Alldatasets were processed with denzo/scalepack or HKL2000 (Otwinowski, Z.,Borek, D., Majewski, W. & Minor, W. Multiparametric scaling ofdiffraction intensities. Acta Crystallogr A 59, 228-34 (2003)).PCSK9/31H4 crystals grew in the C2 space group with unit cell dimensionsa=264.9, b=137.4, c=69.9 Å, β=102.8° and diffract to 2.3 Å resolution.The PCSK9/31H4 structure was solved by molecular replacement with theprogram MOLREP (The CCP4 suite: programs for protein crystallography.Acta Crystallogr D Biol Crystallogr 50, 760-3 (1994) using the PCSK9structure (Piper, D. E. et al. The crystal structure of PCSK9: aregulator of plasma LDL-cholesterol. Structure 15, 545-52 (2007)) as thestarting search model. Keeping the PCSK9 31-692 solution fixed, anantibody variable domain was used as a search model. Keeping the PCSK931-692/antibody variable domain solution fixed, an antibody constantdomain was used as a search model. The complete structure was improvedwith multiple rounds of model building with Quanta and refinement withcnx. (Brunger, A. T. et al. Crystallography & NMR system: A new softwaresuite for macromolecular structure determination. Acta Crystallogr DBiol Crystallogr 54, 905-21 (1998)).

PCSK9/31H4/21B12 crystals grew in the P2₁2₁2 space group with unit celldimensions a=138.7, b=246.2, c=51.3 Å and diffract to 2.8 Å resolution.The PCSK9/31H4/21B12 structure was solved by molecular replacement withthe program MOLREP using the PCSK9 ProCat/31H4 variable domain as thestarting search model. Keeping the PCSK9 ProCat/31H4 variable domainfixed, a search for antibody constant domain was performed. Keeping thePCSK9 ProCat/31H4/21B12 constant domain fixed, an antibody variabledomain was used as a search model. The complete structure was improvedwith multiple rounds of model building with Quanta and refinement withcnx.

PCSK9/EGFa domain crystals grew in the space group P6₅22 with unit celldimensions a=b=70.6, c=321.8 Å and diffract to 2.9 Å resolution. ThePCSK9/EGFa domain structure was solved by molecular replacement with theprogram MOLREP using the PCSK9 ProCat as the starting search model.Analysis of the electron density maps showed clear electron density forthe EGFa domain. The LDLR EGFa domain was fit by hand and the model wasimproved with multiple rounds of model building with Quanta andrefinement with cnx.

Core interaction interface amino acids were determined as being allamino acid residues with at least one atom less than or equal to 5 Åfrom the PCSK9 partner protein. 5 Å was chosen as the core region cutoffdistance to allow for atoms within a van der Waals radius plus apossible water-mediated hydrogen bond. Boundary interaction interfaceamino acids were determined as all amino acid residues with at least oneatom less than or equal to 8 Å from the PCSK9 partner protein but notincluded in the core interaction list. Less than or equal to 8 Å waschosen as the boundary region cutoff distance to allow for the length ofan extended arginine amino acid. Amino acids that met these distancecriteria were calculated with the program PyMOL. (DeLano, W. L. ThePyMOL Molecular Graphics System. (Palo Alto, 2002)).

Example 36 Crystal Structure of PCSK9 and 31A4

The crystal structure of the 31A4/PCSK9 complex was determined.

Expression and Purification of Protein Samples

PCSK9 449TEV (a PCSK9 construct with a TEV protease cleavage siteinserted between residue 449 and 450, numbering according to SEQ ID NO:3) was expressed in baculovirus infected Hi-5 insect cells with anN-terminal honeybee melittin signal peptide followed by a His₆ tag. ThePCSK9 protein was purified by first by nickel affinity chromatography.TEV protease was used to remove the melittin-His₆ tag and cleave thePCSK9 protein between the catalytic domain and V domain. The V domainwas further purified by ion exchange chromatography and size exclusionchromatography. The 31A4 Fab fragment was expressed in E. coli. Thisprotein was purified by nickel affinity chromatography, size exclusionchromatography and ion exchange chromatography.

Complex Formation and Crystallization

The PCSK9 V domain/31A4 complex was made by mixing a 1.5 molar excess ofPCSK9 V domain with 31A4 Fab. The complex was separated from excessPCSK9 V domain by purification on a size exclusion chromatographycolumn. The PCSK9 V domain/31A4 complex crystallized in 1.1 M Succinicacid pH 7, 2% PEG MME 2000.

Data Collection and Structure Determination

The dataset for the PCSK9 V domain/31A4 crystal was collected on aRigaku FR-E x-ray source and processed with denzo/scalepack (Otwinowski,Z., Borek, D., Majewski, W. & Minor, W. Multiparametric scaling ofdiffraction intensities. Acta Crystallogr A 59, 228-34 (2003)).

PCSK9 V domain/31A4 crystals grow in the P2₁2₁2₁ space group with unitcell dimensions a=74.6, b=131.1, c=197.9 Å with two complex moleculesper asymmetric unit, and diffract to 2.2 Å resolution. The PCSK9 Vdomain/31A4 structure was solved by molecular replacement with theprogram MOLREP (CCP4. The CCP4 suite: programs for proteincrystallography. Acta Crystallogr D Biol Crystallogr 50, 760-3 (1994))using the V domain of the PCSK9 structure (Piper, D. E. et al. Thecrystal structure of PCSK9: a regulator of plasma LDL-cholesterol.Structure 15, 545-52 (2007)) as the starting search model. Keeping thePCSK9 450-692 solution fixed, an antibody variable domain was used as asearch model. After initial refinement, the antibody constant domainswere fit by hand. The complete structure was improved with multiplerounds of model building with Quanta and refinement with cnx (Brunger,A. T. et al. Crystallography & NMR system: A new software suite formacromolecular structure determination. Acta Crystallogr D BiolCrystallogr 54, 905-21 (1998)).

Core interaction interface amino acids were determined as being allamino acid residues with at least one atom less than or equal to 5 Åfrom the PCSK9 partner protein. 5 Å was chosen as the core region cutoffdistance to allow for atoms within a van der Waals radius plus apossible water-mediated hydrogen bond. Boundary interaction interfaceamino acids were determined as all amino acid residues with at least oneatom less than or equal to 8 Å from the PCSK9 partner protein but notincluded in the core interaction list. Less than or equal to 8 Å waschosen as the boundary region cutoff distance to allow for the length ofan extended arginine amino acid. Amino acids that met these distancecriteria were calculated with the program PyMOL (DeLano, W. L. The PyMOLMolecular Graphics System. (Palo Alto, 2002)). Distances were calculatedusing the V domain “A” and 31A4 “L1,H1” complex.

The crystal structure of the PCSK9 V domain bound to the Fab fragment of31A4 was determined at 2.2 Å resolution. The depictions of the crystalstructure are provided in FIGS. 21A-21D. FIGS. 21A-21C shows that the31A4 Fab binds to the PCSK9 V domain in the region of subdomains 1 and2.

A model of full length PCSK9 bound the 31A4 Fab was made. The structureof full length PCSK9 was overlaid onto the PCSK9 V domain from thecomplex. A figure of this model is shown in FIG. 21D. The site of theinteraction between the EGFa domain of the LDLR and PCSK9 ishighlighted.

Analysis of the structure shows where this antibody interacts with PCSK9and demonstrated that antibodies that do not bind to the LDLR bindingsurface of PCSK9 can still inhibit the degradation of LDLR that ismediated through PCSK9 (when the results are viewed in combination withExample 40 and 41 below). In addition, analysis of the crystal structureallows for identification of specific amino acids involved in theinteraction between PCSK9 and the 31A4 antibody. Furthermore, the coreand boundary regions of the interface on the PCSK9 surface were alsodetermined. Specific core PCSK9 amino acid residues of the interactioninterface with 31A4 were defined as PCSK9 residues that are within 5 Åof the 31A4 protein. The core residues are T468, R469, M470, A471, T472,R496, R499, E501, A502, Q503, R510, H512, F515, P540, P541, A542, E543,H565, W566, E567, V568, E569, R592, and E593. Boundary PCSK9 amino acidresidues of the interaction interface with 31A4 were defined as PCSK9residues that are 5-8 Å from the 31A4 protein. The boundary residues areas follows: S465, G466, P467, A473, I474, R476, G497, E498, M500, G504,K506, L507, V508, A511, N513, A514, G516, V536, T538, A539, A544, T548,D570, L571, H591, A594, S595, and H597. Amino acid residues nearly orcompletely buried within the PCSK9 protein are highlighted by underline.As noted herein, the numbering references the amino acid positions ofSEQ ID NO: 3 (adjusted as noted herein).

Specific core 31A4 amino acid residues of the interaction interface withPCSK9 were defined as 31A4 residues that are within 5 Å of the PCSK9protein. The core residues for the 31A4 antibody are as follows: HeavyChain: G27, S28, F29, S30, A31, Y32, Y33, E50, N52, H53, R56, D58, K76,G98, Q99, L100, and V101; Light Chain: S31, N32, T33, Y50, S51, N52,N53, Q54, W92, and D94. Boundary 31A4 amino acid residues of theinteraction interface with PCSK9 were defined as 31A4 residues that are5-8 Å from the PCSK9 protein. The boundary residues for 31A4 are asfollows: Heavy Chain: V2, G26, W34, N35, W47, I51, S54, T57, Y59, A96,R97, P102, F103, and D104; Light Chain: S26, S27, N28, G30, V34, N35,R55, P56, K67, V91, D93, S95, N97, G98, and W99.

The crystal structure also displayed the spatial requirements of thisABP in its interaction with PCSK9. As shown in this structure,surprisingly, antibodies that bind to PCSK9 without directly preventingPCSK9's interaction with the LDLR can still inhibit PCSK9's function.

In some embodiments, any antigen binding protein that binds to, covers,or prevents 31A4 from interacting with any of the above residues can beemployed to bind to or neutralize PCSK9. In some embodiments, the ABPbinds to or interacts with at least one of the following PCSK9 (SEQ IDNO: 3) residues: T468, R469, M470, A471, T472, R496, R499, E501, A502,Q503, R510, H512, F515, P540, P541, A542, E543, H565, W566, E567, V568,E569, R592, and E593. In some embodiments, the ABP is within 5 angstromsof one or more of the above residues. In some embodiments, the ABP bindsto or interacts with at least one of the following PCSK9 (SEQ ID NO: 3)residues: S465, G466, P467, A473, I474, R476, G497, E498, M500, G504,K506, L507, V508, A511, N513, A514, G516, V536, T538, A539, A544, T548,D570, L571, H591, A594, S595, and H597. In some embodiments, the ABP is5 to 8 angstroms from one or more of the above residues. In someembodiments, the ABP interacts, blocks, or is within 8 angstroms of 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45,or 50 of the above residues.

The coordinates for the crystal structures discussed in the aboveExamples are are presented in Table 35.1 (full length PCSK9 and 31H4),Table 35.2 (PCSK9 and EGFa), Table 35.3 (PCSK9, 31H4, and 21B12), andTable 35.4 (PCSK9 and 31A4). Antigen binding proteins and molecules thatinteract with the relevant areas or residues of the structure of PCSK9(including those areas or residues within 15, 15-8,8,8-5, 5, or fewerangstroms from where EGFa, or the antibodies, interact with PCSK9)depicted in the figures and/or their corresponding positions on thestructures from the coordinates are also contemplated.

The antibodies that are described in the coordinates were raised in E.coli and thus possess some minor amino acid differences from the fullyhuman antibodies. The first residue in the variable region was aglutamic acid instead of a glutamine for the heavy and light chains of21B12 and for the light chain for 31H4. In addition to the differencesin the sequence of variable region, there were also some differences inthe constant region of the antibodies described by the coordinates(again due to the fact that the antibody was raised in E. coli). FIG. 22highlights (via underlining shading, or bold) the differences betweenthe constant regions of the 21B12, 31H4, and 31A4 Fabs (raised in E.coli) when compared to SEQ ID NOs: 156, and 155. For 21B12 31H4, and31A4, the light chain constant sequence is similar to human lambda (SEQID NO: 156). The underlined glycine residue is an insertion betweenwhere the 21B12 and 31H4 variable sequences stop and the lambda sequencestarts.

For both 21B12 and 31H4, the heavy chain constant is similar to humanIgG4 (SEQ ID NO: 155). The highlighted differences in FIG. 22 are shownin Table 36.1:

TABLE 36.1 Crystal SEQ ID NO: 155 S C K R G E G S Q K I T N D K R P S

In regard to 31A4, while it also has the same distinctions noted above,there are three additional differences. As shown in FIG. 22, there aretwo additional amino acids at the start, which comes from incompleteprocessing of the signal peptide in E. coli expression. In addition,there is one additional substitution in the 31A4 heavy chain constantregion when compared to SEQ ID NO: 155, which is the adjustment of a L(in SEQ ID NO: 155) to a H. Finally, 31A4 does have a glutamine as theinitial amino acid of the Fab, rather than the adjustment to glutamicacid noted above for 21B12 and 31H4.

For all three antibodies, the end of the heavy chain (boxed in darkgrey) differs as well, but the amino acids are not ordered in thestructure so they do not appear in the coordinates. As will beappreciated by one of skill in the art, his-tags are not a required partof the ABP and should not be considered as part of the ABP's sequence,unless explicitly called out by reference to a specific SEQ ID NO thatincludes a histidine tag and a statement that the ABP sequence “includesthe Histidine tag.”

Example 37 Epitope Mapping—Binning

An alternative set of binning experiments was conducted in addition tothe set in Example 10. As in Example 10, ABPs that compete with eachother can be thought of as binding to the same site on the target and incommon parlance are said to “bin” together.

A modification of the Multiplexed Binning method described by Jia, et al(J. Immunological Methods, 288 (2004) 91-98) was used. Individual beadcodes of streptavidin-coated Luminex beads was incubated in 100 ul 0.5ug/ml biotinylated monovalent mouse-anti-human IgG capture antibody (BDPharmingen, #555785) for 1 hour at room temperature in the dark, thenwashed 3× with PBSA, phosphate buffered saline (PBS) plus 1% bovineserum albumin (BSA). Each bead code was separately incubated with 100 ul2 ug/ml anti-PCSK9 antibody (Coating Antibody) for 1 hour then washed 3×with PBSA. The beads were pooled then dispensed to a 96-well filterplate (Millipore, #MSBVN1250). 100 ul of 2 ug/ml purified PCSK9 proteinwas added to half the wells. Buffer was added to the other half ascontrol. The reaction was incubated for 1 hour then washed. 100 ul of a2 ug/ml anti-PCSK9 antibody (Detection Ab) was added to all the wells,incubated for 1 hour then washed. An irrelevant human-IgG (Jackson,#009-000-003) was run as another control. 20 ul PE-conjugated monovalentmouse-anti-human IgG (BD Pharmingen, #555787) was added to each well andincubated for 1 hour then washed. Beads were resuspended in 100 ul PBSAand a minimum of 100 events/bead code were collected on the BioPlexinstrument (BioRad).

Median Fluorescent Intensity (MFI) of the antibody pair without PCSK9was subtracted from signal of the corresponding reaction containingPCSK9. For the antibody pair to be considered bound simultaneously, andtherefore in different bins, the subtracted signal had to be greaterthan 3 times the signal of the antibody competing with itself and the 3times the signal of the antibody competing with the irrelevant antibody.

The data from the above is depicted in FIGS. 23A-23D. The ABPs fell intofive bins. The shaded boxes indicate ABPs that can bind simultaneouslyto PCSK9. The nonshaded boxes indicate those ABPs that compete with eachother for binding. A summary of the results is shown in Table 37.1.

TABLE 37.1 BIN 1 BIN 2 BIN 3 BIN 4 BIN 5 01A12.2 27B2.1 16F12.1 11G1.530A4.1 03B6.1 27B2.5 22E2.1 03C4.1 13B5.1 09C9.1 12H11.1 27A6.1 13H1.117C2.1 28B12.1 31A4.1 21B12.2 28D6.1 31B12.1 23G1.1 31G11.1 25G4.131H4.1 26E10.1 08A1.2 11H4.1 08A3.1 11H8.1 11F1.1 19H9.2 26H5.1 27E7.127H5.1 30B9.1 02B5.1 23B5.1 27B2.6 09H6.1

Bins 1 (competes with ABP 21B12) and 3 (competes with 31H4) areexclusive of each other; bin 2 competes with bins 1 and 3; and Bin 4does not compete with bins 1 and 3. Bin 5, in this example, is presentedas a “catch all” bin to describe those ABPs that do not fit into theother bins. Thus, the above identified ABPs in each of the binds arerepresentative of different types of epitope locations on PCSK9, some ofwhich overlap with each other.

As will be appreciated by one of skill in the art, if the reference ABPprevents the binding of the probe ABP then the antibodies are said to bein the same bin. The order in which the ABPs are employed can beimportant. If ABP A is employed as the reference ABP and blocks thebinding of ABP B the converse is not always true: ABP B used as thereference ABP will not necessarily block ABP A. There are a number offactors in play here: the binding of an ABP can cause conformationalchanges in the target which prevent the binding of the second ABP, orepitopes which overlap but do not completely occlude each other mayallow for the second ABP to still have enough high-affinity interactionswith the target to allow binding. ABPs with a much higher affinity mayhave a greater ability to bump a blocking ABP out of the way. Ingeneral, if competition is observed in either order the ABPs are said tobin together, and if both ABPs can block each other then it is likelythat the epitopes overlap more completely.

Example 38 Epitope Mapping—Western Blot

The present example demonstrates whether or not the epitopes for theexamined ABPs were linear or conformational. Denaturing reducing anddenaturing non-reducing western blots were run to determine whichantibodies have a conformational epitope. Antibodies that bind to adenaturing reducing western blot have a linear epitope and are notconformational. The results are presented in FIG. 24A and FIG. 24B. Forthe blot, 0.5 ug/lane of purified full-length human PCSK9 was run on a4-12% NuPAGE Bis-Tris gel and MES SDS Running Buffer. 1 ug/ml anti-PCSK9antibodies, except 0.5 ug/ml 31G11, were used to probe the blot. 1:5000donkey-anti-human-IR700 secondary was used and read on a LiCORinstrument. Antibody 13H1 bound to a linear epitope on the pro-domain ofPCSK9. All other antibodies displayed results that were consistent withconformational epitopes. These gels split apart the pro-domain from therest of the protein, and the pro domain ran at about 15 kDa. Inaddition, 3C4 and 31A4 appeared to bind to conformational epitopes whichwere preserved by disulfide bonds, as these antibodies bound to PCSK-9under denaturing conditions where the disulfide bonds had been preserved(left) but reducing the samples (right) eliminated binding.

Example 39 Epitope Mapping—Arginine/Glutamic Acid Scanning

Representative ABPs from each bin (from Example 37) were selected forfurther epitope analysis. An arginine/glutamic acid-scanning strategywas performed for mapping ABP binding to PCSK9. By way of background,this method determines if a residue is part of the structural epitope,meaning those residues in the antigen which contact or are buried by theantibody. Arginine and glutamic acid sidechains are charged and bulkyand can disrupt antibody binding even if the mutated residue is notdirectly involved in antibody binding.

Residue Selection

The crystal structure of PCSK9 was used to select the residues to bemutated for epitope mapping. The method used to choose residues tomutate involved both computational mechanisms and interactive structureanalysis. The PCSK9 structure contained gaps of missing residues and wasmissing 30 amino acids in the N— (i.e., the signal sequence) and 10amino acids in the C-termini. The internal missing residues were modeledonto the structure, but the N- and C-terminal missing residues were not.The solvent exposure ratio for each residue was calculated: the surfacearea of each residue in the context of the protein (SA1) was divided bythe surface area of the residue in a trimer with flanking glycines (SA2)with a conserved backbone structure. Residues with solvent exposureratio greater than 10% (R10) were selected as well as the 40 missingterminal residues. From these, prolines and glycines with positive Φangles were excluded to reduce the possibility of misfolding. The numberof residues to be mutated in the V domain was reduced by using a solventexposure ratio of 37% along with visual inspection of the entire proteinto bring the total number of mutations to 285. Various orientations ofthe surface of PCSK9 with these various classes identifies are shown inFIG. 25A-25F. In these figures, lightest gray denotes areas that werenot selected or were deselected. darker gray denotes those residuesselected).

Cloning and Expression

Once the residues to be altered were identified, the various residueswere altered. Human PCSK9 was cloned into the pTT5 vector with aC-terminal Flag-His tag. Mutants were made from this original constructby site-directed mutagenesis using a QuikChange II kit from Stratagene.Sense and anti-sense oligonucleotides used for mutagenesis were designedusing Amgen's MutaGenie software. All PCSK9 constructs were expressed intransiently-transfected 293-6E cells in 24-well plates and re-rackedinto three 96-well plates with a non-mutated PCSK9 control (wild-type,WT) in each plate. Expression levels and integrity of the recombinantproteins in conditioned media were checked by Western blot. Of the 285mutants originally selected, 41 failed in cloning or expression. 244mutants were used for epitope mapping. An alignment of the PCSK9 parentsequence and a representative PCSK9 sequence with the 244 mutatedresidues is shown in FIG. 26. Separate constructs were made containing asingle mutation. For the purposes of the epitope sequences and theepitope based inventions involving changes in binding, the sequences areprovided in reference to SEQ ID NO: 1 and/or SEQ ID NO: 303. Thesequences in FIG. 26 were the sequences used for the present bindingepitope studies. One of skill in the art will appreciate that thepresent results apply to other PCSK9 variants disclosed herein as well(e.g., SEQ ID NO: 1 and 3, as well as the other allelic variants).

Five antibodies, a representative of each bin, were chosen for fineepitope mapping. They were 21B12, 31H4, 12H11, 31A4, 3C4. Allconformational epitope antibodies. Three, 21B12, 31H4, and 31A4 werealso crystallized with PCSK9, as described above.

Structural and Functional Epitopes

Epitopes can be further defined as structural or functional. Functionalepitopes are generally a subset of the structural epitopes and havethose residues that directly contribute to the affinity of theinteraction (e.g. hydrogen bonds, ionic interactions). Structuralepitopes can be thought of as the patch of the target which is coveredby the antibody.

The scanning mutagenesis employed was an arginine and glutamic acidscan. These two sidechains were chosen due to their large steric bulkand their charge, which allows mutations that occur in the structuralepitope to have a greater effect on antibody binding. Arginine wasgenerally employed except when the WT reside was arginine, and in thesecases the residue was mutated to glutamic acid to switch the charge.

For the purpose of epitope mapping, a bead-based multiplexed assay wasused to measure antibody binding to PCSK9 and PCSK9 mutantssimultaneously. Antibody binding to mutants was then compared to itsbinding to the wild-type in the same well. The variants were split intothree groups: Group 1: 81 variants+2 wt controls+1 negative control+1other PCSK9 supernatant; Group 2: 81 variants+2 wt controls+2 negativecontrols; and Group 3: 82 variants+2 wt control+1 negative control.

The assay was run as follows: 85 sets of color-coded strepavidin-coatedLumAvidin beads (Luminex) were bound with biotinylated anti-pentaHisantibody (Qiagen, #1019225) for 1 hour at room temperature (RT) thenwashed three times in PBS, 1% BSA, 0.1% Tween 20. Each color-coded beadset was then allowed to bind to a PCSK9 mutant, wild-type, or negativecontrol in 150 ul supernatant overnight at 4° C.

The color-coded bead sets, each associated to a specific protein, werewashed and pooled. At this point, there were 3 pools of 85 bead sets,one pool for each group of mutants and controls. The beads from eachpool were aliquoted to 24 wells (3 columns) of a 96-well filter plate(Millipore, #MSBVN1250). 100 ul of anti-PCSK9 antibodies in 4-folddilutions were added to nine columns for triplicate points and incubatedfor 1 hour at RT and washed. 100 ul of 1:200 dilution phycoerythrin(PE)-conjugated anti-human IgG Fc (Jackson Immunoresearch, #109-116-170)was added to each well and incubated for 1 hour at RT and washed.

Beads were resuspended in 1% BSA in PBS, shaken for 10 mins and read onthe BioPlex instrument (Bio-Rad). The instrument identifies each bead byits color-code thereby identifying the specific protein associated withthe color code. At the same time, it measures the amount of antibodybound to the beads by fluorescence intensity of the PE dye. Antibodybinding to each mutant can then be compared directly its binding to thewild type in the same pool. IL-17R chimera E was used as a negativecontrol. A summary of all of the mutants examined is shown in Table 39.1(with reference to the sequence numbering used in FIGS. 1A and 26).

TABLE 39.1 1 2 3 4 5 6 7 8 9 10 11 12 A WT PCSK9 Y8R E18R P26R A38R T56RA70R H83R E102R L128R D145R B Q1R E9R E19R E27R K39R H57R Q71R V84RL105R E129R S148R C E2R E10R D20R G29R D40R L58R A73R H86R K106R R130Epcsk9 supe test D D3R L11R G21R T30R L44R Q60R R74E K95R H109R T132RIL17R chimera E E E4R V12R L22R T31R T47R E62R R75E S97R D111R D139R WTPCSK9 F D5R A14R A23R A32R K53R R63E Y77R G98R A121R E140R G G6R L15RE24R T33R E54R R66E L78R D99R S123R Y141R H D7R S17R A25R H35R E55R R67EL82R L101R W126R Q142R A WT PCSK9 M171R E181R Q189R K213R R242E G251RL294R L321R Q352R E380R B L149R V172R D182R A190R G214R K243R G262RA311R E336R M368R R384E C S158R T173R G183R S191R S216R S244R R265EQ312R D337R S371R IL17R chimera E D Q160R D174R T184R K192R R221E Q245RA269R D313R D344R A372R IL17R chimera E E S161R E176R R185E S195R Q226RL246R Q272R Q314R T347R E373R WT PCSK9 F D162R N177R F186R H196R K228RV247R R276E T317R F349R E375R G R164E V178R H187R R207E T230R Q248RA277R L318R V350R T377R H E167R E180R R188E D208R F240R V250R R289ET320R S351R L378R A WT PCSK9 N395R V405R W423R R446E E513R Q525R Q554RQ589R S632R A641R B I386R E396R N409R Q424R D450R A514R E537R N556RQ591R T633R R650E C H387R A397R A413R A433R A472R S515R V538R K579RA595R T634R R652E D F388R W398R S417R H434R F485R M516R E539R V580RE597R G635R IL17R chimera E E A390R E401R T418R T438R G486R R519E L541RK581R E598R S636R WT PCSK9 F K391R D402R H419R R439E E488R H521R H544RE582R V620R T637R G D392R Q403R G420R M440R N503R H523R V548R H583RR629E S638R H V393R R404E A421R T442R T508R Q524R R552E G584R V631RE639R

Bead Variability Study

Before running the epitope mapping binding assay, a validationexperiment was conducted to assess the “bead region” to “bead region”(B-B) variability. In the validation experiment, all beads wereconjugated with the same wild type control protein. Therefore, thedifference between beads regions was due to purely B-B variance and wasnot confounded by difference between wild type and mutant proteins. Thetitration of antibody was run with twelve replications in differentwells.

The objective of this statistical analysis was to estimate the B-Bvariability of the estimated EC50 of binding curves. The estimated B-Bstandard deviation (SD) was then used to build the EC50 confidenceintervals of wild type and mutant proteins during curve comparisonexperiments.

A four-parameter logistic model was fitted to the binding data for eachbead region. The resulting file, containing curve quality control (QC)results and parameter estimates for top (max), bottom (min), Hillslope(slope), and natural log of EC50 (xmid) of the curves, was used as theraw data for the analysis. B-B variability for each parameter was thenestimated by fitting mixed effect model using SAS PROC MIXED procedure.Only curves with “good” QC status were included in the analysis. Thefinal mixed effect model included only residual (i.e. individual beadregions) as random effect. Least squares means (LS-mean) for eachparameter were estimated by the mixed effect model as well. B-B SD wascalculated by taking square root of B-B variance. Fold change betweenLS-mean+2SD and LS-mean−2SD, which represent approximately upper andlower 97.5 percentile of the population, was also calculated. Theresults are displayed in Table 39.2

TABLE 39.2 Least square mean and bead-to-bead variance estimations AssayID parname Ls Mean B-B Variance −2SD +2SD Fold Change* PCSK9 max 15000997719 13002.3 16997.7 1.3 PCSK9 min 162.09 1919.66 74.5 249.7 3.4 PCSK9slope 0.8549 0.000599 0.8 0.9 1.1 PCSK9 xmid 3.1715 0.002098 3.1 3.3 1.2*xmid is natural log of the EC50. Fold change for xmid was convertedback to original scale.

Identifying Residues in the Structural Epitope

A residue was considered part of the structural epitope (a “hit”) whenmutating it to arginine or glutamic acid alters antibody binding. Thisis seen as a shift in the EC50 or a reduction of maximum signal comparedto antibody binding to wild type. Statistical analyses of antibodybinding curves to wild type and mutants were used to identifystatistically significant EC50 shifts. The analysis takes intoconsideration variation in the assay and curve fitting.

Hit Identification Based on EC50 Comparison

The EC50 and Bmax values were generated from a Weighted 4-ParameterLogistical model fitted to the binding data using S-PLUS with VarPowersoftware (Insightful Corporation, Seattle Wash.). The EC50s of themutant binding curves and wild type binding curves were compared.Statistically significant differences were identified as hits forfurther consideration. The curves with “nofit” or “badfit” flags wereexcluded from the analysis.

The Variations in EC50 Estimates

Two sources of variations were considered in the comparison of EC50estimates, variation from the curve fit and the bead-bead variation.Wild types and mutants were linked to different beads, hence theirdifference are confounded with the bead-bead difference (describedabove). The curve fit variation was estimated by the standard error ofthe log EC50 estimates. Bead-bead variation was experimentallydetermined using an experiment where wild type controls were linked toeach one of the beads (described above). The bead variation in EC50estimates of wild type binding curve from this experiment was used toestimate the bead-bead variation in the actual epitope mappingexperiment.

Testing for EC50 Shift Between Mutants and Wild Type

The comparisons of two EC50s (in log scale) was conducted usingStudent's t-test. The t-statistic was calculated as the ratio betweendelta (the absolute differences between EC50 estimates) and the standarddeviation of delta. The variance of delta was estimated by the sum ofthe three components, variance estimate of EC50 for mutant and wild typecurves in the nonlinear regression and two times the bead-bead varianceestimated from a separate experiment. The multiple of two for thebead-bead variance was due to the assumption that both mutant and wildtype beads had the same variance. The degree of freedom of the standarddeviation of delta was calculated using the Satterthwaite's (1946)approximation. Individual p-values and confidence intervals (95% and99%) were derived based on Student's t distribution for each comparison.In the case of multiple wild type controls, a conservative approach wastaken by picking the wild type control that was most similar to themutant, i.e., picking the ones with the largest p-values.

Multiplicity adjustments were important to control the false positive(s)while conducting a large number of tests simultaneously. Two forms ofmultiplicity adjustment were implemented for this analysis: family wiseerror (FWE) control and false discovery rate (FDR) control. The FWEapproach controls the probability that one or more hits are not real;FDR approach controls the expected proportion of false positive amongthe selected hits. The former approach is more conservative and lesspowerful than the latter one. There are many methods available for bothapproaches, for this analysis, the Hochberg's (1988) method for FWEanalysis and Benjamini-Hochberg's (1995) FDR method for FDR analysiswere selected. Adjusted p-values for both approaches were calculated.

Results

EC50 Shift

Mutations whose EC50 is significantly different from wild type, e.g.,having a False Discovery Rate adjusted p-value for the whole assay of0.01 or less, were considered part of the structural epitope. All thehits also had a Familywise type I error rate adjusted p-value for eachantibody of less than 0.01 except residue R185E for antibody 31H4 whichhad an FWE adjusted p-value per antibody of 0.0109. The residues in thestructural epitope of the various antibodies determined by EC50 shiftare shown in Table 39.3 (point mutations are with reference to SEQ IDNO: 1 and 303)

TABLE 39.3 FDR.Adjusted. FWE.Adjusted. Antibody Mutation Pval By.Pval.Low99 Low95 FoldChange High95 High99 RawPval 21B12 D208R 0.0000 0.00000.3628 0.3844 0.4602 0.5509 0.5837 0.0000 21B12 R207E 0.0000 0.00001.7148 1.8488 2.3191 2.9090 3.1364 0.0000 31H4 R185E 0.0024 0.01091.2444 1.3525 1.7421 2.2439 2.4388 0.0000 31A4 E513R 0.0001 0.00031.4764 1.6219 2.1560 2.8660 3.1485 0.0000 31A4 E539R 0.0000 0.00001.6014 1.7461 2.2726 2.9578 3.2252 0.0000 31A4 R439E 0.0000 0.00003.1565 3.6501 5.5738 8.5113 9.8420 0.0000 31A4 V538R 0.0004 0.00131.4225 1.5700 2.1142 2.8471 3.1423 0.0000 12H11 A390R 0.0000 0.00011.4140 1.5286 1.9389 2.4594 2.6588 0.0000 12H11 A413R 0.0009 0.00281.2840 1.3891 1.7653 2.2434 2.4269 0.0000 12H11 S351R 0.0009 0.00281.2513 1.3444 1.6761 2.0896 2.2452 0.0000 12H11 T132R 0.0000 0.00011.3476 1.4392 1.7631 2.1599 2.3068 0.0000 3C4 E582R 0.0016 0.0069 1.35231.5025 2.0642 2.8359 3.1509 0.0000

Maximum Signal Reduction

The percent maximum signal was calculated using the maximum signal fromthe curve fitting (BmaxPerWT) and raw data point (RawMaxPerWT).Mutations that reduced the antibody binding maximum signal by ≧70% ascompared to wild type signal or that reduced the signal of one antibodycompared to other antibodies by >50% when all other antibodies are atleast 40% of wild type were considered hits and part of the epitope.Table 39.4 displays the residues that are in the structural epitope(italics) as determined by reduction of maximum signal.

TABLE 39.4 antibody Mutants BmaxPerWT RawMaxPerWT 21B12 A311R 141.6388139.7010 31H4 A311R 145.2189 147.8244 31A4 A311R 103.4377 96.2214 12H11A311R 14.9600 3C4 A311R 129.0460 131.2060 21B12 D162R 7.0520 31H4 D162R108.8308 112.4904 31A4 D162R 98.8873 95.9268 12H11 D162R 94.6280 97.49283C4 D162R 101.4281 100.1586 21B12 D313R 45.8356 45.0011 31H4 D313R45.6242 44.9706 31A4 D313R 47.9728 44.7741 12H11 D313R 16.1811 18.42623C4 D313R 58.5269 57.6032 21B12 D337R 61.9070 62.2852 31H4 D337R 63.160464.1029 31A4 D337R 62.9124 59.4852 12H11 D337R 10.8443 3C4 D337R 73.032673.9961 21B12 E129R 139.9772 138.9671 31H4 E129R 141.6792 139.1764 31A4E129R 77.3005 74.8946 12H11 E129R 28.6398 29.3751 3C4 E129R 85.770185.7802 21B12 E167R 15.1082 31H4 E167R 127.4479 128.2698 31A4 E167R115.3403 112.6951 12H11 E167R 111.0979 109.6813 3C4 E167R 109.3223108.7864 21B12 H521R 133.8480 133.9791 31H4 H521R 130.2068 128.4879 31A4H521R 124.5091 129.3218 12H11 H521R 130.7979 134.4355 3C4 H521R 22.107721B12 Q554R 125.9594 125.2103 31H4 Q554R 122.2045 128.7304 31A4 Q554R113.6769 121.3369 12H11 Q554R 116.1789 118.4170 3C4 Q554R 31.8416 21B12R164E 17.3807 19.8505 31H4 R164E 97.8218 99.6673 31A4 R164E 98.259596.3352 12H11 R164E 88.0067 89.8807 3C4 R164E 105.0589 105.7286 21B12R519E 139.4598 141.2949 31H4 R519E 135.5609 140.0000 31A4 R519E 134.2303137.1110 12H11 R519E 135.4755 137.0824 3C4 R519E 44.0091 21B12 S123R87.6431 88.1356 31H4 S123R 85.5312 84.7668 31A4 S123R 68.4371 66.613112H11 S123R 20.8560 20.6910 3C4 S123R 73.6475 71.5959 (Point mutationsare with reference to SEQ ID NO: 1 and FIG. 26).

Table 39.5 displays a summary of all of the hits for the variousantibodies.

TABLE 39.5 EC50 shift hits Bmax shift hits 21B12 31H4 31A4 12H11 3C421B12 31H4 31A4 12H11 3C4 R207E R185E R439E T132R E582R D162R S123RR519E D208R* E513R S351R R164E E129R H521R V538R A390R E167R A311R Q554RE539R A413R D313R D337R *decreases EC50

To further examine how these residues form part of or all of therelevant epitopes, the above noted positions were mapped onto variouscrystal structure models, the results are shown in FIG. 27A through 27E.FIG. 27A depicts the 21B12 epitope hits, as mapped onto a crystalstructure of PCSK9 with the 21B12 antibody. The structure identifiesPCSK9 residues as follows: light gray indicates those residues that werenot mutated (with the exception of those residues that are explicitlyindicated on the structure) and darker gray indicates those residuesmutated (a minority of which failed to express). Residues that areexplicitly indicated were tested (regardless of the shading indicated onthe figure) and resulted in a significant change in EC50 and/or Bmax Theepitope hits were based on Bmax shift. In this figure, 31H4 is behind 21B12.

FIG. 27B depicts the 31H4 epitope hits, as mapped onto a crystalstructure of PCSK9 with 31H4 and 21B12 antibodies. The structureidentifies PCSK9 residues as follows: light gray indicates thoseresidues that were not mutated (with the exception of those residuesthat are explicitly indicated on the structure) and darker grayindicates those residues mutated (a minority of which failed toexpress). Residues that are explicitly indicated were tested (regardlessof the shading indicated on the figure) and resulted in a significantchange in EC50 and/or Bmax. The epitope hits were based on the EC50shift.

FIG. 27C depicts the 31A4 epitope hits, as mapped onto a crystalstructure of PCSK9 with 31H4 and 21B12 antibodies. The structureidentifies PCSK9 residues as follows: light gray indicates thoseresidues that were not mutated (with the exception of those residuesthat are explicitly indicated on the structure) and darker grayindicates those residues mutated (a minority of which failed toexpress). Residues that are explicitly indicated were tested (regardlessof the shading indicated on the figure) and resulted in a significantchange in EC50 and/or Bmax. The epitope hits were based on the EC50shift. 31A4 antibody is known to bind to the V-domain of PCSK9, whichappears consistent with the results presented in FIG. 27C.

FIG. 27D depicts the 12H11 epitope hits, as mapped onto the crystalstructure of PCSK9 with 31H4 and 21B12 antibodies. The structureidentifies PCSK9 residues as follows: light gray indicates thoseresidues that were not mutated (with the exception of those residuesthat are explicitly indicated on the structure) and darker grayindicates those residues mutated (a minority of which failed toexpress). Residues that are explicitly indicated were tested (regardlessof the shading indicated on the figure) and resulted in a significantchange in EC50 and/or Bmax. 12H11 competes with 21B12 and 31H4 in thebinning assay described above.

FIG. 27E depicts the 3C4 epitope hits, as mapped onto the crystalstructure of PCSK9 with 31H4 and 21B12 antibodies. The structureidentifies PCSK9 residues as follows: light gray indicates thoseresidues that were not mutated (with the exception of those residuesthat are explicitly indicated on the structure) and darker grayindicates those residues mutated (a minority of which failed toexpress). Residues that are explicitly indicated were tested (regardlessof the shading indicated on the figure) and resulted in a significantchange in EC50 and/or Bmax.

3C4 does not compete with 21B12 and 31H4 in the binning assay. 3C4 bindsto the V-domain in the domain binding assay (see results from Example40, FIGS. 28A and 28B).

While there were approximately a dozen mutants that could have beenexpected to have an effect on binding (based upon the crystalstructure), the present experiment demonstrated that, surprisingly, theydid not. As will be appreciated by one of skill in the art, the resultspresented above are in good agreement with the crystal structures andPCSK-9's binding of these antibodies. This demonstrates that theprovided structural and corresponding functional data adequatelyidentifies the key residues and areas of interaction of the neutralizingABPs and PCSK9. Thus, variants of the ABPs that possess the ability tobind to the above noted areas are adequately provided by the presentdescription.

As will be appreciated by one of skill in the art, while the B-max dropand EC50 shift hits can be considered manifestations of the samephenomenon, strictly speaking, a B-max drop alone does not reflect aloss of affinity per se but, rather, the destruction of some percentageof the epitope of an antibody. Although there is no overlap in the hitsdetermined by B-max and EC50, mutations with a strong affect on bindingmay not allow for the generation of a useful binding curve and hence, noEC50 can be determined for such variants.

As will be appreciated by one of skill in the art, ABPs in the same bin(with the exception of bin 5, which as noted above, is a general catchall bin) likely bind to overlapping sites on the target protein. Assuch, the above epitopes and relevant residues can generally be extendedto all such ABPs in the same bin.

To further examine the above results in regard to ABP 31H4, positionE181R, which, according to the above crystal structure, was predicted tointeract with R185 to form part of the surface that interacts with theABP, was also altered (E181R). The results, while not statisticallysignificant on their own, were, when combined with the crystalstructure, demonstrative of 31H4 interacting with E181R (data notshown). Thus, position 181 also appears to form part of the epitope forthe 31H4 ABP.

As noted above, the above binding data and epitope characterizationreferences a PCSK9 sequence (SEQ ID NO: 1) that does not include thefirst 30 amino acids of PCSK9. Thus, the numbering system of thisprotein fragment, and the SEQ ID NO:s that refer to this fragment, areshifted by 30 amino acids compared to the data and experiments that useda full length PCSK9 numbering system (such as that used in the crystalstudy data described above). Thus, to compare these results, an extra 30amino acids should be added to the positions in each of the aboveepitope mapping results. For example, position 207 of SEQ ID NO: 1 (orSEQ ID NO: 303), correlates to position 237 of SEQ ID NO: 3 (the fulllength sequence, and the numbering system used throughout the rest ofthe specification). Table 39.6 outlines how the above noted positions,which reference SEQ ID NO: 1 (and/or SEQ ID NO: 303) correlate with SEQID NO: 3 (which includes the signal sequence).

TABLE 39.6 AMINO ACID POSITION IN AMINO ACID POSITION IN SEQ SEQ ID NO:1 (EPITOPE DATA) ID NO: 3 (EPITOPE DATA) 207 237 208 238 185 215 181 211439 469 513 543 538 568 539 569 132 162 351 381 390 420 413 443 582 612162 192 164 194 167 197 123 153 129 159 311 341 313 343 337 367 519 549521 551 554 584

Thus, those embodiments described herein with reference to SEQ ID NO: 1can also be described, by their above noted corresponding position withreference to SEQ ID NO: 3.

Example 40 PCSK9 Domain Binding Assay

The present example examined where on PCSK9 the various ABPs bound.

Clear, 96 well maxisorp plates (Nunc) were coated overnight with 2 ug/mlof various anti-PCSK9 antibodies diluted in PBS. Plates were washedthoroughly with PBS/0.05% Tween-20 and then blocked for two hours with3% BSA/PBS. After washing, plates were incubated for two hours witheither full length PCSK9 (aa 31-692 SEQ ID NO: 3, procat PCSK9 (aa31-449 SEQ ID NO: 3) or v-domain PCSK9 (aa 450-692 of SEQ ID NO: 3)diluted in general assay diluent (Immunochemistry Technologies, LLC).Plates were washed and a rabbit polyclonal biotinylated anti-PCSK9antibody (D8774), which recognizes the procat and v-domain as well asfull-length PCSK9, was added at 1 ug/ml (in 1% BSA/PBS). Boundfull-length, procat or v-domain PCSK9 was detected by incubation withneutravidin-HRP (Thermo Scientific) at 200 ng/ml (in 1% BSA/PBS)followed by TMB substrate (KPL) and absorbance measurement at 650 nm.The results, presented in FIGS. 28A and 28B, demonstrate the ability ofthe various ABS to bind to various parts of PCSK9. As shown in FIG. 28B,ABP 31A4 binds to the V domain of PCSK9.

Example 41 Neutralizing, Non-Competitive Antigen Binding Proteins

The present example demonstrates how to identify and characterize anantigen binding protein that is non-competitive with LDLR for bindingwith PCSK9, but is still neutralizing towards PCSK9 activity. In otherwords, such an antigen binding protein will not block PCSK9 from bindingto LDLR, but will prevent or reduce PCSK9 mediated LDLR degradation.

Clear, 384 well plates (Costar) were coated with 2 ug/ml of goatanti-LDL receptor antibody (R&D Systems) diluted in buffer A (100 mMsodium cacodylate, pH 7.4). Plates were washed thoroughly with buffer Aand then blocked for 2 hours with buffer B (1% milk in buffer A). Afterwashing, plates were incubated for 1.5 hours with 0.4 ug/ml of LDLreceptor (R&D Systems) diluted in buffer C (buffer B supplemented with10 mM CaCl₂). Concurrent with this incubation, 20 ng/ml of biotinylatedD374Y PCSK9 was incubated with 100 ng/ml of antibody diluted in buffer Aor buffer A alone (control). The LDL receptor containing plates werewashed and the biotinylated D374Y PCSK9/antibody mixture was transferredto them and incubated for 1 hour at room temperature. Binding of thebiotinylated D374Y to the LDL receptor was detected by incubation withstreptavidin-HRP (Biosource) at 500 ng/ml in buffer C followed by TMBsubstrate (KPL). The signal was quenched with 1N HCl and the absorbanceread at 450 nm. The results are presented in FIG. 28C, which shows thatwhile ABP 31H4 inhibits LDLR binding, ABP 31A4 does not inhibit LDLRbinding to PCSK9. In combination with the results from Example 40 andshown in FIGS. 28A and 28B, it is clear that 31A4 ABP binds to the Vdomain of PCSK9 and does not block the interaction of PCSK9 with LDLR.

Next, the Ability of ABP 31A4 to serve as a neutralizing ABP was furtherconfirmed via a cell LDL uptake assay (as described in the examplesabove). The results of this LDL uptake assay are presented in FIG. 28D.As shown in FIG. 28D, ABP 31A4 displays significant PCSK9 neutralizingability. Thus, in light of Example 40 and the present results, it isclear that ABPs can bind to PCSK9 without blocking the PCSK9 and LDLRbinding interaction, while still being useful as neutralizing PCSK9ABPs.

INCORPORATION BY REFERENCE

All references cited herein, including patents, patent applications,papers, text books, and the like, and the references cited therein, tothe extent that they are not already, are hereby incorporated herein byreference in their entirety. To the extent that any of the definitionsor terms provided in the references incorporated by reference differfrom the terms and discussion provided herein, the present terms anddefinitions control.

EQUIVALENTS

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The foregoingdescription and examples detail certain preferred embodiments of theinvention and describe the best mode contemplated by the inventors. Itwill be appreciated, however, that no matter how detailed the foregoingmay appear in text, the invention may be practiced in many ways and theinvention should be construed in accordance with the appended claims andany equivalents thereof.

1. An isolated neutralizing antigen binding protein that binds to aPCSK9 protein comprising the amino acid sequence of SEQ ID NO: 1,wherein the neutralizing antigen binding protein comprises: a heavychain polypeptide comprising the following complementarity determiningregions (CDRs): a heavy chain CDR1 that is a CDR1 in SEQ ID NO: 67; aheavy chain CDR2 that is a CDR2 in SEQ ID NO: 67; a heavy chain CDR3that is a CDR3 in SEQ ID NO: 67; and a light chain polypeptidecomprising the following CDRs: a light chain CDR1 that is a CDR1 in SEQID NO: 12; a light chain CDR2 that a CDR2 in SEQ ID NO: 12; and a lightchain CDR3 that is a CDR3 in SEQ ID NO:
 12. 2. (canceled)
 3. Theisolated neutralizing antigen binding protein of claim 1, wherein theantigen binding protein is a LDLR competitive neutralizing antigenbinding protein. 4-28. (canceled)
 29. A pharmaceutical compositioncomprising at least one antigen binding protein according to claim 1,and a pharmaceutically acceptable excipient. 30-36. (canceled)
 37. Theisolated neutralizing antigen binding protein of claim 1, wherein theheavy chain polypeptide comprises all three of the following amino acidsequences: SEQ ID NO: 247, SEQ ID NO: 256, and SEQ ID NO:
 265. 38. Theisolated neutralizing antigen binding protein of claim 1, wherein thelight chain polypeptide comprises all three of the following amino acidsequences: SEQ ID NO: 222, SEQ ID NO: 229, and SEQ ID NO:
 238. 39. Theisolated neutralizing antigen binding protein of claim 38, wherein theheavy chain polypeptide comprises all three of the following amino acidsequences: SEQ ID NO: 247, SEQ ID NO: 256, and SEQ ID NO:
 265. 40. Theisolated neutralizing antigen binding protein of claim 1, wherein thelight chain polypeptide comprises: a CDR1 comprising SEQ ID NO: 222, aCDR2 comprising SEQ ID NO: 229, and a CDR3 comprising SEQ ID NO: 238.41. The isolated neutralizing antigen binding protein of claim 1,wherein the heavy chain polypeptide comprises: a CDR1 comprising SEQ IDNO: 247, a CDR2 comprising SEQ ID NO: 256, and a CDR3 comprising SEQ IDNO:
 265. 42. The isolated neutralizing antigen binding protein of claim41, wherein the light chain polypeptide comprises: a CDR1 comprising SEQID NO: 222, a CDR2 comprising SEQ ID NO: 229, and a CDR3 comprising SEQID NO:
 238. 43. The isolated neutralizing antigen binding protein ofclaim 1, wherein the heavy chain polypeptide comprises SEQ ID NO: 67 andthe light chain polypeptide comprises SEQ ID NO:
 12. 44. The isolatedneutralizing antigen binding protein of claim 1, wherein each CDR isdefined in accordance with the Kabat definition, the Chothia definition,the combination of the Kabat definition and the Chothia definition, theAbM definition, or the contact definition of CDR.
 45. The isolatedneutralizing antigen binding protein of claim 44, wherein each CDR isdefined in accordance with the CDR definition of Kabat.
 46. The isolatedneutralizing antigen binding protein of claim 44, wherein each CDR isdefined in accordance with the CDR definition of Chothia.
 47. Theisolated neutralizing antigen binding protein of claim 44, wherein eachCDR is defined in accordance with the combination of the Kabatdefinition and the Chothia definition.
 48. An isolated neutralizinghuman monoclonal antibody that binds to a PCSK9 protein comprising: aheavy chain polypeptide comprising the following complementaritydetermining regions (CDRs): a heavy chain CDR1 that is a CDR1 in SEQ IDNO: 67; a heavy chain CDR2 that is a CDR2 in SEQ ID NO: 67; a heavychain CDR3 that is a CDR3 in SEQ ID NO: 67; and a light chainpolypeptide comprising the following CDRs: a light chain CDR1 that is aCDR1 in SEQ ID NO: 12; a light chain CDR2 that a CDR2 in SEQ ID NO: 12;and a light chain CDR3 that is a CDR3 in SEQ ID NO: 12, wherein each CDRis defined in accordance with the CDR definition of Kabat.
 49. Theisolated neutralizing human monoclonal antibody of claim 48, wherein theheavy chain polypeptide comprises all three of the following amino acidsequences: SEQ ID NO: 247, SEQ ID NO: 256, and SEQ ID NO:
 265. 50. Theisolated neutralizing human monoclonal antibody of claim 48, wherein thelight chain polypeptide comprises all three of the following sequences:SEQ ID NO: 222, SEQ ID NO: 229, and SEQ ID NO:
 238. 51. The isolatedneutralizing human monoclonal antibody of claim 50, wherein the heavychain polypeptide comprises all three of the following sequences: SEQ IDNO: 247, SEQ ID NO: 256, and SEQ ID NO:
 265. 52. The isolatedneutralizing human monoclonal antibody of claim 48, wherein the heavychain polypeptide comprises SEQ ID NO: 67 and the light chainpolypeptide comprises SEQ ID NO:
 12. 53. An isolated neutralizing humanmonoclonal antibody that binds to a PCSK9 protein comprising: a heavychain polypeptide comprising: the amino acid sequence of SEQ ID NO: 247;the amino acid sequence of SEQ ID NO: 256; and the amino acid sequenceof SEQ ID NO: 265; and a light chain polypeptide comprising: the aminoacid sequence of SEQ ID NO: 222; the amino acid sequence of SEQ ID NO:229; and the amino acid sequence of SEQ ID NO:
 238. 54. An isolatedneutralizing human monoclonal antibody that binds to a PCSK9 proteincomprising: a heavy chain polypeptide comprising the followingcomplementarity determining regions (CDRs): a heavy chain CDR1 that is aCDR1 in SEQ ID NO: 247; a heavy chain CDR2 that is a CDR2 in SEQ ID NO:256; a heavy chain CDR3 that is a CDR3 in SEQ ID NO: 265; and a lightchain polypeptide comprising the following CDRs: a light chain CDR1 thatis a CDR1 in SEQ ID NO: 222; a light chain CDR2 that is a CDR2 in SEQ IDNO: 229; and a light chain CDR3 that is a CDR3 in SEQ ID NO:
 238. 55. Anisolated neutralizing human monoclonal antibody that binds to a PCSK9protein comprising: a heavy chain polypeptide comprising the followingcomplementarity determining regions (CDRs): a heavy chain CDR1 that is aCDR1 in SEQ ID NO: 67; a heavy chain CDR2 that is a CDR2 in SEQ ID NO:67; a heavy chain CDR3 that is a CDR3 in SEQ ID NO: 67, wherein theheavy chain polypeptide comprises: the amino acid sequence of SEQ ID NO:247; the amino acid sequence of SEQ ID NO: 256; and the amino acidsequence of SEQ ID NO: 265; and a light chain polypeptide comprising thefollowing CDRs: a light chain CDR1 that is a CDR1 in SEQ ID NO: 12; alight chain CDR2 that a CDR2 in SEQ ID NO: 12; and a light chain CDR3that is a CDR3 in SEQ ID NO: 12, wherein the light chain polypeptidecomprises: the amino acid sequence of SEQ ID NO: 222; the amino acidsequence of SEQ ID NO: 229; and the amino acid sequence of SEQ ID NO:238.